Phytochemical Screening

Introduction A phytochemical is a natural bioactive compound found in plant foods that works with nutrients and dietary fiber to protect against disease. Research suggests that phytochemicals, working together with nutrients found in fruits, vegetables and nuts, may help slow the aging process and reduce the risk of many diseases, including cancer, heart disease, stroke, high blood pressure, cataracts, osteoporosis, and urinary tract infections. Pronounced "fight-o-chemicals," phytochemicals fight to protect your health. They can have complementary and overlapping mechanisms of action in the body, including antioxidant effects, modulation of detoxification enzymes, stimulation of the immune system, modulation of hormone metabolism, and antibacterial and antiviral effect. "Phyto" is a Greek word that means plant and phytochemicals are usually related to plant pigments. So, fruits and vegetables that are bright colors — yellow, orange, red, green, blue, and purple — generally contain the most phytochemicals and the most nutrients. You can benefit from all of the phytochemicals and nutrients found in plant foods by eating 5-9 servings of fruits and vegetables a day and eating more whole grains, soy and nuts. More than 900 different phytochemicals have been found in plant foods and more will be discovered. These protective plant compounds are an emerging area of nutrition and health, with new research reported everyday. Remember, to get your Phytos eat 5-9 servings of colorful fruits and vegetables every day! The following charts provide a description of the most well researched phytochemicals and some of the fruits and vegetables they are found in. Complete phytochemical analysis has not been done on most fruits and vegetables. USDA will conduct phytochemical analysis on approximately 100 of the most frequently eaten fruits and vegetables during 2000-2001. Our charts will be updated as more phytochemical research becomes available. Current research suggests that most fruits and vegetables contain phytochemicals and that many fruits and vegetables contain a wide variety of Phytochemicals.

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History of phytochemical Only a few years ago, the term "phytochemical" was barely known. But doctors, nutritionists, and other health care practitioners have long advocated a low-fat diet that includes a variety of fruits, vegetables, legumes, and whole grains. Historically, cultures that consume such a diet have lower rates of certain cancers and heart disease. Since the passage of the Dietary Supplement Health and Education Act (DSHEA) in the United States in 1994, a large number of phytochemicals are being sold as dietary supplements.

Evidence of phytochemical It has become a widely accepted notion that a diet rich in fruits, vegetables, legumes, and grains reduces the risk of cancer, heart disease, and other illnesses. But only recently have researchers begun to try to learn the effects of specific phytochemicals contain those foods. Much of the evidence so far has come from observations of cultures whose diets consist mainly of plant sources, and which seem to have lower rates of certain types of cancer and heart disease. For instance, the relatively low rates of breast and endometrial cancers in some Asian cultures are credited at least in part to dietary habits. These cancers are much more common in the United States, possibly because the typical American diet is higher in fat and lower in fruits, vegetables, legumes, and grains. Because of the number of phytochemicals and the complexity of the chemical processes they are involved in, researchers face a challenging task in trying to determine which phytochemicals in foods may fight cancer and other diseases, which may have no effect, and which may even be harmful. Many studies have looked at the relationship between cancer risk and eating fruits and vegetables, legumes, and whole grains. Most of the evidence indicates that eating large proportions of these foods seems to lower the risk of some cancers and other illnesses.

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Some of the links between individual phytochemicals and cancer risk found in studies in the lab are very compelling and make a very strong case for the need for further research. So far, however, none of the findings is conclusive. It is still uncertain which of the many phytochemicals in fruits and vegetables actively helps the body fight disease. Researchers have also shown much interest in phytochemical supplements. Some lab studies in cell cultures and animals have shown that certain phytochemicals have some activity against cancer cells or tumors. But at this time there have been no strong studies in humans showing that any phytochemical supplement can prevent or treat cancer. Until conclusive research findings emerge, health care professionals advise a balanced diet with an emphasis on fruits, vegetables, legumes, and whole grains. The interaction between certain phytochemicals and the other compounds in foods is not well understood, but it is unlikely that any single compound offers the best protection against cancer. A balanced diet that includes 5 or more servings a day of fruits and vegetables along with foods from a variety of other plant sources such as nuts, seeds, whole grain cereals, and beans is likely to be more effective in reducing cancer risk than eating one particular phytochemical in large amounts. Carotenoids are the pigments responsible for the colors of many red, green, yellow and orange fruits and vegetables. Carotenoids are a large family of phytochemicals which include alpha-carotene, beta-carotene, lutein, lycopene, cryptoxanthin, canthaxanthin, zeaxanthin, and others. Carotenoids protect the body by decreasing risk of heart disease, stroke, blindness, and certain types of cancer. They may also help to slow the aging process, reduce complications associated with diabetes, and improve lung function. Fruits and vegetables that are dark green, yellow, orange or red contain carotenoids.The following information describe four of the carotenoids.

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Source of phytochemical Beta-Carotene Beta-Carotene may help to slow the aging process, reduce the risk of certain types of cancer, improve lung function, and reduce complications associated with diabetes. Beta-carotene is found in yellow-orange fruits and vegetables such as mangoes, cantaloupe, apricots, papaya, kiwifruit, carrots, pumpkins, sweet potatoes, and winter squash, and green vegetables, such as broccoli, spinach, and kale. Lutein Lutein is essential for maintaining proper vision as we age. It has been shown to reduce the risk of cataracts and macular degeneration, the leading causes of blindness in older people and may help reduce the risk of certain types of cancer. Kale, spinach and collard greens contain the most lutein of any fruit or vegetable. Other sources of lutein include kiwifruit, broccoli, collard greens, brussels sprouts, swiss chard, and romaine lettuce. Lycopene Diets rich in lycopene have been shown to reduce the risk of prostate cancer and heart disease. Lycopene is found in red fruits and vegetables such as tomatoes and cooked tomato products, red peppers, pink grapefruit, watermelon. Zeaxanthin Zeaxanthin may help to prevent macular degeneration, which is the leading cause of visual impairment in people over 50. It may also help to prevent certain types of cancer. Corn, spinach, winter squash, and egg yolks contain zeaxanthin. Flavonoids Flavonoids are another large family of protective phytochemicals found in fruits and vegetables. Flavonoids, also called bioflavonoids, act as antioxidants. Antioxidants neutralize or inactivate highly unstable and extremely reactive molecules, called free radicals that attack the cells of our body every day. Free radical damage is believed to contribute to a variety of health problems, including cancer, heart disease and aging. There are many different types of flavonoids and each appears to have protective health effects. Some of the better known flavonoids include resveratrol, anthocyanins, quercetin, hesperidin, tangeritin, kaempferol, myricetin, and apigenin. Flavonoids are found in a variety of foods, such as oranges, kiwifruit, grapefruit, tangerines, berries, apples, 4

red grapes, red wine, broccoli, onions, and green tea. The five primary flavonoids found in fruits and vegetables are: Resveratrol Resveratrol may reduce the risk of heart disease, cancer, blood clots and stroke. Red grapes, red grape juice, and red wine contain resveratrol. Anthocyanins Anthocyanins, which are particularly high in blueberries, have been shown to protect against the signs of aging. In one study, elderly rats that ate the equivalent of a half-cup of blueberries daily for eight weeks improved balance, coordination, and short-term memory. Scientists think these results may apply to humans as well. Anthocyanins in blueberries and cranberries have also been shown to help prevent urinary tract infections. Blueberries, cherries, strawberries, kiwifruit, and plums contain anthocyanins. Quercetins Quercetins may reduce inflammation associated with allergies, inhibit the growth of head and neck cancers, and protect the lungs from the harmful effects of pollutants and cigarette smoke. Apples, pears, cherries, grapes, onions, kale, broccoli, leaf lettuce, garlic, green tea, and red wine contain quercetins. Hesperidin Hesperidin is a flavonoid that may protect against heart disease. Hesperidin is found in citrus fruits and fruit juices, such as oranges and orange juice, grapefruit and grapefruit juice, tangerines, lemons, limes, mandarins, and tangelos. Tangeritin Tangeritin may help prevent cancers of the head and neck. Tangeritin is found in citrus fruits and their juices. Phenolic Compounds Phenolic compounds may reduce the risk of heart disease and certain types of cancer. Phenolic compounds may be found in berries, prunes, red grapes and red grape juice, kiwifruit, currants, apples and apple juice, and tomatoes. Ellagic Acid Ellagic acid is a phenolic compound that may reduce the risk of certain types of cancer and decrease cholesterol levels. Ellagic acid is found in red grapes, kiwifruit, blueberries, raspberries, strawberries, blackberries, and currants. Sulphoraphane Sulphoraphane is in a class of phytochemicals called isothiocyanates. Sulphoraphane may reduce the risk of colon cancer. Cruciferous vegetables such as broccoli sprouts, broccoli, 5

cauliflower, kale, Brussels sprouts, cabbage, bok choy, collard greens, turnips and turnip greens contain sulphoraphane. Limonene Limonene is in a class of phytochemicals called mono-terpenes. It is found in the rinds and the edible white membranes of citrus fruits, such as oranges, grapefruit, tangerines, lemons and limes. Limonene may help to protect the lungs and reduce the risk of certain types of cancer. Indoles This family of phytochemicals may reduce the risk of certain types of cancer, including breast cancer. Indoles are found in cruciferous vegetables, such as broccoli, cauliflower, kale, brussels sprouts, cabbage, bok choy, collard greens, watercress, and turnips and turnip greens. Allium Compounds Allium compounds may reduce the risk of certain types of cancer and lower cholesterol and blood pressure. Garlic, onions, chives, leeks, and scallions contain allium compounds.

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Apples and apple juice contain phenolic compounds which may protect against heart disease. Apricots (fresh and dried) contain beta-carotene which may help slow the aging process, reduce the risk of certain types of cancer, improve lung function, and reduce complications associated with diabetes. Blackberries contain ellagic acid which may reduce the risk of certain forms of cancer and decrease cholesterol levels. Blueberries contain anthocyanins which may protect against the effects of aging. Blueberries also contain ellagic acid which may reduce the risk of certain forms of cancer and decrease cholesterol levels. Bok Choy contains a variety of phytochemicals including sulphoraphane and indoles. Broccoli contains many different phytochemicals including sulphoraphane, indoles, beta-carotene, lutein, and quercetins. These phytochemicals may help slow the aging process, reduce the risk of certain types of cancer, improve lung function, protect against macular degeneration and cataracts, reduce inflammation associated with allergies, and reduce complications associated with diabetes. 6

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Broccoli sprouts contain sulphoraphane which may reduce the risk of certain types of cancer. Brussel sprouts contain a variety of phytochemicals including sulphoraphane and indoles. These phytochemicals may reduce the risk of certain types of cancer. Cabbage contains a variety of phytochemicals including sulphoraphane and indoles. These phytochemicals may reduce the risk of certain types of cancer. Cantaloupe contains beta-carotene which may help slow the aging process, reduce the risk of certain types of cancer, improve lung function, and reduce complications associated with diabetes.

Carrots contain beta-carotene which may help slow the aging process, reduce the risk of certain types of cancer, improve lung function, and reduce complications associated with diabetes. Cauliflower contains a variety of phytochemicals including sulphoraphane and indoles. These phytochemicals may reduce the risk of certain types of cancer. Cherries contain anthocyanins which may protect against the signs of aging. Cherries also contain quercetins which may reduce inflammation associated with allergies, inhibit the growth of head and neck tumors, and protect the lungs from the harmful effects of pollutants and cigarette smoke. Chives contain allium compounds that may reduce the risk of certain forms of cancer and lower cholesterol and blood pressure. Citrus fruits, such as oranges, grapefruits, and tangerines contain hesperidin and tangeritin which act as antioxidants to reduce the risk of heart disease and various types of cancer. Citrus fruits also contain limonene which may protect the lungs. Collard greens contain lutein which may reduce the risk of cataracts and macular degeneration. Collard greens also contain indoles and sulphoraphane which may help decrease the risk of certain types of cancer. Corn contains zeaxanthin which may help to prevent macular degeneration, which is the leading cause of visual impairment in people over 50. Currants contain ellagic acid which may reduce the risk of certain forms of cancer and decrease cholesterol levels. Garlic contains allium compounds which may reduce the risk of certain forms of cancers and lower cholesterol levels and blood pressure. Garlic also contains quercetins which may reduce inflammation associated with allergies, inhibit the growth of head 7

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and neck tumors, and protect the lungs from the harmful effects of pollutants and cigarette smoke. Kale contains a variety of phytochemicals including beta carotene which may help slow the aging process, reduce the risk of certain types of cancer, improve lung function, and reduce complications associated with diabetes and lutein which may reduce the risk of cataracts and macular degeneration. Kale also contains indoles and sulphoraphane which may help decrease cancer risk and quercetins which may reduce inflammation associated with allergies, inhibit the growth of head and neck tumors, and protect the lungs from the harmful effects of pollutants and cigarette smoke. Kiwifruit contains a variety of phytochemicals, including betacarotene, lutein, anthocyanins, and ellagic acid. These phytochemicals may reduce the risk of heart disease, certain types of cancer, cataracts, and macular degeneration. Leaf Lettuce contains quercetins which may reduce inflammation associated with allergies, inhibit the growth of head and neck tumors, and protect the lungs from the harmful effects of pollutants and cigarette smoke. Leeks contain allium compounds which reduce the risk of certain forms of cancer and may lower cholesterol levels and blood pressure. Mangoes contain beta-carotene which may help slow the aging process, reduce the risk of certain forms of cancer, improve lung function, and reduce complications associated with diabetes. Onions contain allium compounds which may reduce the risk of certain forms of cancer and lower cholesterol levels and blood pressure. Onions also contain quercetins which may reduce inflammation associated with allergies, inhibit the growth of head and neck tumors, and protect the lungs from the harmful effects of pollutants and cigarette smoke. Papaya contain beta-carotene which may help slow the aging process, reduce the risk of certain forms of cancer, improve lung function, and reduce complications associated with diabetes. Pears contain quercetins which may reduce inflammation associated with allergies, inhibit the growth of head and neck tumors, and protect the lungs from the harmful effects of pollutants and cigarette smoke. Pink grapefruit contains lycopene which may decrease risk for prostate cancer and heart disease. Pink grapefruit also contains hesperidin and tangeritin which act as antioxidants to reduce the 8

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risk of heart disease and various types of cancer as well as limonene which may protect the lungs. Plums contain anthocyanins which may help protect against the signs of aging. Prunes contain phenolic compounds which act as antioxidants that may prevent the loss of long-term memory and learning ability. Pumpkins contain beta-carotene which may help slow the aging process, reduce the risk of certain types of cancer, improve lung function, and reduce complications associated with diabetes. Raisins contain phenolic compounds that may act as powerful antioxidants to help slow the aging process. Raspberries contain ellagic acid which may reduce the risk of certain forms of cancer and decrease cholesterol levels. Red grapes and grape juice contain resveratrol and ellagic acid which may lower the risk of heart disease and certain forms of cancer.. Red grapes also contain quercetins which may reduce inflammation associated with allergies, inhibit the growth of head and neck tumors, and protect the lungs from the harmful effects of pollutants and cigarette smoke. Red peppers contain lycopene which reduce the risk of prostate cancer and heart disease. Romaine lettuce contains lutein which may reduce the risk of cataracts and macular degeneration, the leading causes of visual impairment in people over 50. Scallions contain allium compounds which may reduce the risk of certain forms of cancer and lower cholesterol levels and blood pressure. Spinach contains beta-carotene which may help slow the aging process, reduce the risk of certain types of cancer, improve lung function, and reduce complications associated with diabetes. Spinach also contains lutein and zeaxanthin which may help prevent blindness. People who eat lots of spinach have a decreased risk of cataracts and macular degeneration, the leading causes of visual impairment in people over 50. Strawberries contain anthocyanins which may protect against the effects of aging. Strawberries also contain ellagic acid which may reduce the risk of certain forms of cancer and decrease cholesterol levels. Sweet potatoes contain beta-carotene which may help slow the aging process, reduce the risk of certain types of cancer, improve lung function, and reduce complications associated with diabetes. 9

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Swiss chard contains lutein which may reduce the risk of cataracts and macular degeneration. Swiss chard also contains indoles and sulphoraphane which may help decrease the risk of certain types of cancer. Tomatoes and cooked tomato products contain lycopene which may decrease risk for prostate cancer and heart disease. Tomato products such as ketchup, tomato juice, and spaghetti sauce are some excellent sources of lycopene. Turnips contain indoles and sulphoraphane which may help decrease the risk of certain types of cancer. Watercress contains indoles and sulphoraphane which may help decrease the risk of certain types of cancer. Watermeloncontains lycopene which may decrease risk for prostate cancer and heart disease. Winter squash contains beta-carotene which may help slow the aging process, reduce the risk of certain forms of cancer, improve lung function, and reduce complications associated with diabetes. Winter squash also contains zeaxanthin which may help to prevent macular degeneration, which is the leading cause of visual impairment in people over 50.

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Phytochemical Definition & chemistry Phytochemicals are non-nutritive plant chemicals that have protective or disease preventive properties. There are more than thousand known phytochemicals. It is well-known that plant produces these chemicals to protect itself but recent research demonstrates that they can protect humans against diseases. Some of the well-known phytochemicals are lycopene in tomatoes, Isoflavones in soy and Flavonoids in fruits. They are not essential nutrients and are not required by the human body for sustaining life.

Phytochemicals are chemicals found in plants. Plant sterols, flavonoids (FLAV'oh-noidz), and sulfur-containing compounds are three classes of micronutrients found in fruits and vegetables. These compounds may be important in reducing the risk of atherosclerosis (ath"er-o-skleh-RO'sis), which is the buildup of fatty deposits in artery walls. Within these categories are many possible compounds, most of which aren't well described and whose modes of action aren't established. Many other plant products may also be linked to the atherosclerotic process, such as antioxidant vitamins, phyto estrogens and trace minerals. These plant micronutrients will clearly be the topic of future research. As work continues on all these compounds, other unrecognized components in plants will be identified that may have promise in reducing risk of cardiovascular disease.

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Mechanism of action of Phytochemicals

There are many phytochemicals and each works differently. These are some possible actions: •

Antioxidant - Most phytochemicals have antioxidant activity and protect our cells against oxidative damage and reduce the risk of developing certain types of cancer. Phytochemicals with antioxidant activity: allyl sulfides (onions, leeks, garlic), carotenoids (fruits, carrots), flavonoids (fruits, vegetables), polyphenols (tea, grapes).

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Hormonal action – Isoflavones, found in soy, imitate human estrogens and help to reduce menopausal symptoms and osteoporosis.

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Interference with DNA replication - Saponins found in beans interfere with the replication of cell DNA, thereby preventing the multiplication of cancer cells. Capsaicin, found in hot peppers, protects DNA from carcinogens.

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Anti-bacterial effect - The phytochemical allicin from garlic has anti-bacterial properties.

Physical action - Some phytochemicals bind physically to cell walls thereby preventing the adhesion of pathogens to human cell walls. Proanthocyanidins are responsible for the anti-adhesion properties of cranberry. Consumption of cranberries will reduce the risk of urinary tract infections and will improve dental health.

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Phytochemical are useful in complication These products are sold as a dietary supplement in the United States. Unlike drugs (which must be tested before being allowed to be sold), the companies that make supplements are not required to prove to the Food and Drug Administration that their supplements are safe or effective, as long as they don't claim the supplements can prevent, treat, or cure any specific disease. Some such products may not contain the amount of the herb or substance that is written on the label, and some may include other substances (contaminants). Actual amounts per dose may vary between brands or even between different batches of the same brand. Most such supplements have not been tested to find out if they interact with medicines, foods, or other herbs and supplements. Even though some reports of interactions and harmful effects may be published, full studies of interactions and effects are not often available. Because of these limitations, any information on ill effects an Phytochemicals in the amounts consumed in a healthy diet are likely to be helpful and are unlikely to cause any major problems. Some people assume that because phytochemical supplements come from "natural" sources, they must be safe and free from side effects, but this is not always true. It is important to note that, especially when taken in large amounts, many of them have side effects and possible interactions with some drugs. Some of these interactions may be dangerous. Before taking a phytochemical in supplement form, consider talking to your doctor and pharmacist tobe sure it won’t interact harmfully with other medicines or herbs you may be taking.

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LIST OF PHYTOCHEMICALS Alkaloids • • •

Caffeine Theobromine Theophylline

Anthocyanins

Carotenoids • •

Beta-Carotene Lycopene

Coumestans Flavan-3-Oil • • • • • • • • • •

Epicatechin Hesperidin Kaempferol Naringin Nobiletin Proanthocyanidins Quercetin Resveratrol Rutin Tangeretin

Hydroxycinnamic Acids • • • •

Chicoric acid Coumarin Ferulic acid Scopoletin

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Isoflavones •

Genistein

Lignans •

Silymarin

Monoterpenes •

Limonene

Organosulfides • • • • •

Allicin Glutathione Indole-3-Carbinol Isothiocyanates Sulforaphane

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Damnacanthal Digoxin Phytic acid

Phenolic Acids • • • • •

Capsaicin Ellagic Acid Gallic acid Rosmarinic acid Tannic Acid

Phytosterols •

Beta-Sitosterol

Saponins Triterpenoids •

Ursolic acid

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SCREENING Definition Screening is the process of separation and isolation of active principle from plant sources.

Screening are helpful-

To get lead for “Discovery of new therapeutic agents”. To find “New sources” for economic material. To help expand “CHEMOTAXONOMY”. To produce “Semisynthetic” derivatives.

For this purpose, following 3 essential steps are prescribed* Selection of plant. * Phytochemicals screening. * Phytopharmacological evaluation.

PHYTOCHEMICAL SCREENING APPROACHES Ultimately, the goal in surveying plants for biologically active or rnedicinally useful compounds should be to isolate the one or more constituents responsible for a particular activity. Hence with the selection of a specific plant for phytochemical investigation either on the basis of one or more approaches set forth under phytopharmacologic Approaches, or through some other avenue, phytochemical screening techniques can be a valuable aid. Certain investigators feel that an initial selection of investigational plants should be made not on evidence that extracts elicit a particular and interesting biological activity, but rather on the basis that certain chemicals are present in the plant, relatives of which can usually be associated with biological activity. Thus, some investigators will select initially only alkaloid-containing plants for study on the premises that (a) normally exert some type of pharmacologic activity usually on the center nerves system but not always so; m(b) the greatest majority of natural products used in medicine today are alkaloidal in 16

nature;(c) tests for the presence of these compounds in plants are simple, can be conducted rapidly, and are reasoanably reliable, and(d)because of their chemical nature, alkaloids are more easily manipulated making extraction and isolation less of a problem . In addition, economics , as well as other factor associated with biological testing, often force the investigation to pursue a phytochemical group other then alkaloids be selected for investigation say the flavonols , the diversity of expected biological activities can be enormous. Willaman has surveyed the literature and has found that at least natural flavonoids are known, occurring in some families, genera, and species of plants . Also, some different pharmacologic or biological activities have been reported for one or more of flavonoids . More recently, horhammer and wagner have reviewed the same area, and these numbers are therefore to be increased . Also, orzechowski has considered the role of flavonids as therapeutic agent. Along similar lines, the coumarins have been repoterd to exert some 31 different biological effert , and according to soine , their full range of farmacologic activities is not apprericiated by most investigators. Other examples pointing out the complexity of expected biological effects for any one category of phytoconstituents could of course be made.In any event publication representing the phytochemical screening approach for out wigh those following phytopharmacologic avenues not only numbers of report but in representation of total plant examined. Since the number of chemical categories of plant constitutes is great and each is capable of eliciting biological activity no attempt will be made in this to be all enclusive. This section of the review will be restricted to some consideration of phytochemical screening methodology followed by discussions of those categories of phytoconstituents Which have been represented in major published surveys of screening programs. These will include: alkaloids. Glycorides as a general class (heteroside) sapgrams. (steroid and triterpenoid).sterols. cardiac glycosides. Cyanogenetic glycosides. Isothiocyanate glycosides. cyanogenetic glycosdes, isothiocyanate glycosides, anthraquinones, flavonoids and related compounds. Surveys which have decn condussed along with the general metehodology involved. The examples to be coted are intended to be representative of each class and are not recant to include all available published data. 17

General considerations A method for use in phytochemical screening should be (a) Simple, (b) Rapid, (c) Designed for a minimum of equipment (d) Reasonably selective for the class of compounds under study (e)Quantitative in so far as having a knowledge of the lower limit of detection is concered, and if possible (f) Should give additional information as to the presence or absence of specific members of the group being evaluated. Most published procedures adhere to criteria. (a) through (d) but few are designed to provide the information included in (e) and (f). In fact, certain procedure cannot be duplicated because of insufficient details included in some report. For example,Arthur and cheung in a phytochemical survey of Hong kong plants, screened 332 species for alkaloids. They equated the precipitates observed following the additional of standard alkaloid precipitating reagents to result obtained by addition the same reagents to standard solution of 1:100, 1:500, 1:2500, and 1;10,000quinine sulfate. It is implied that water was the solvent. However , the solubility of quinine sulfate is stated to be 1gm.in 810 ml. of water. Along similar lines, will have used the cyanidin test for the detection of the alfa-benzofurane nucleus as indicative of the presence of flavanoids. They compare a test result color with a similar color produced by a 0.1% solution of rutin and equate it as a (+) reaction. Their extraction solvent is 95% ethanol (but fresh plant inaterial was often extracted which would decrease this pcrcentage considerably), and rutin is stated to be only slightly soluble in ethanol and soluble about l Gm. in 8 L. of water. We find that maximum solubility of rutin at room temperature is about 0.02% for both 80 and 95% ethanol. Webb, using a field method, estimated alkaloid precipitates with reagents on a + to ++++ basis but used no reference for comparison . IIe also states,.. on the other hand, while the method may yield a percentage of ‘false positives; it has never failed to 18

detect species with’’ alkaloids . If the initial field test did indeed fail to detect alkaloids, perhaps because of a low concentration in the plant, how could it be determined that the test was infallible when only field test positives species were colleted for more specific laboratory examination .

Fundamental considerationOne of the most important and fundamental consideration in designing a phytochemical screening produce is the selection of proper extraction solvent.It is often difficult to follow general or expected solubility rule for a given class of phytoconsitutents scince there are often substance of unknown Character present in crude plant extracts that affect solubility. For example, woo has repoted that effect of saponin in plant extracts on the solubility of certain normally insoluble compounds using selected solvent. Apparently saponin acts as a wetting agent to enhance the formation of micelles ;thus an increase in solubility of certain constituents is effected. This phenomena has been noted through the use of synthetic detergents to enhance the solubility, and thus extract ability of alkaloids from cinchona . Since saponins, or other similar surface-active agents, do not occur universally in plants, prediction of general solubilities for a class of phytoconstituents ppt. a major problem. In our laboratory n-hexane-soluble extractives from catharanthus lanceus were found to be rich in alkaloids. Subsequent isolation of individual alkaloids from the crude mixture proved them to be totally insoluble in n-hexane. Presumably the alkaloids occur in the plant, at least in this instance dissolved in some lipid material, the latter being soluble in n-hexane. No solution is offered for these problemes involving solubility except tosay that extract residues should always be examined with a variety of solvents to determine whether solubility phenomena have occurred. Even though a great many problems are presented by the diverse methodology utilized by investigatiors in phytochemical screening, much useful information can be derived from published studies. Positive test result are usually clear cut and on the other hand, must be carefully weighed in terms of being due to real absence of the test material in the sample being evaluated, or to the methodology employed.

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Pharmavegetables, their ethnomedicinal properties and phytochemical screening of plant extracts.

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Plant Species Common Ethnomedicinal Name Property Abelmoschus Lagikway Emmenagogue; laxative manihot (L.)

Amaranthus spinosus L.

Kadyapa

Amaranthus tricolor L. Amaranthus tricolor L. Amaranthus viridis L.

Dalaura Pula’t Dilaw

Broussonetia luzonica (Blanco) var. luzonica

Salugim

Kulitis

Cayratia sp. (?) Yagini

Centella asiatica

TakipKuhol

Colubrina asiatica (L.)

Kabatete

Corchorus capsularis (L.) Cordia dichotoma Forst. F.

Saluyot ligaw Anonang

Eclipta alba (L.) HigisHassk. Manok

Phytochemicals in the Plant Extract Flavonoid, unsat. sterol & triterpene, steroid Medik glycoside, tannin & phenol Antiinflammatory, Flavonoid, unsat. sterol & antussive, diuretic, triterpene, cyanogenic febrifuge, kidney, tonic, glycoside, tannin & lactagogue, laxative phenol Antidysentery, antitussive Flavonoid, unsat. sterol & triterpene, tannin & phenol Antiinflammatory, antitumor, antitussive

Flavonoid, unsat. sterol & triterpene, cyanogenic glycoside, tannin & phenol Alkaloid, flavonoid, unsat. sterol & triterpene, steroid glycoside, cyanogenic glycoside, tannin & phenol Flavonoid, unsat. sterol & triterpene, tannin & phenol Antiaging, antidysentery, Alkaloid, flavonoid, unsat. anti-inflammatory, sterol & triterpene, steroid antitumor, diuretic, glycoside, cyanogenic immunostimulant, liver glycoside,saponin, tonic anthraquinone Alkaloid, flavonoid, unsat. Alkaloid, flavonoid, unsat. sterol & triterpene, sterol & triterpene, steroid saponin, anthraquinone, glycoside, cyanogenic, tannin & phenol glycoside, & saponin, tannin phenol Demulcent Antidysentery, diuretic, liver tonic

Antianemia, immunostimulant, 21 vermifuge

Alkaloid, flavonoid, unsat. sterol & triterpene, steroid glycoside, cyanogenic glycoside, saponin, tannin & phenol Alkaloid, flavonoid, unsat. sterol & triterpene, steroid glycoside, cyanogenic glycoside,saponin, tannin

Alkaloid screening Prior to a consideration of screening plant material for alkaloids . it would seem in order to define the term “alkaloids “ as used in this review ;however the nature of the word . It self precludes any thing more then a vague definition. Any one familiar with alkaloids surely has a knowledge of their character but seldom can one give as acceptable definition. Most authorities agree that chemical botanical and pharmacologic implications most be reflected an one acceptable definition. Hegnauers suggestion that; Alkaloids are more or less toxic substances which act primarily on the central nervous system have a basic character contain heterocyclic nitrogen and are synthesized in plants from amino acids or their immediate derivatives. In most eases they are of limited distribution in the plant kingdom. Seems as acceptable as any. For purposes of this discussion we will utilize Hegnaures concept except. Of course, we cannot be concerned with the site of mechanism of synthesis , thus compounds such as aliphatic nitrogenous bases amides and the amino acids themselves will not be considered as alkaloids, Estimates for the distribution of alkaloids in vascular plants have been placed as high as 15-20%, although this figure appears some what high with respect to data derived from several extensive phytochemical screening programs Will have screened more then 4000 species of plants and report a distribution of about 10% alkaloids. Webb in his experience with some 1700 species indicates alkaloids occurrence to be about 14% whereas the smith kline & French survey found that about 10% of 25000 species screened were positive for alkaloids . since a few of these undoubtedly will be determined through future studies to be false positive alkaloid containing species, 9-10%seems to be the more logical estimate representing alkaloids yielding plant species. Alkaloids are widely distributed in the plant kingdom although certain groups have been shown to be characteristically devoid of them excellent essays on this subject have been published by willaman and Schubert and by Webb.

22

ALKALOID DETECTION Since alkaloids usually occur in plants as their water-soluble salts, some workers believe that extraction with acidulated water can result in a crude extract which can be tested directly with one or more standard alkaloid ppt. reagents. Other workers feel that the presence in such an extract of materials that are capable of giving false-positive alkaloids test necessitate a purification procedure before valid result can be obtained. This is usually accomplished by the addition of base and subsequent extraction with waterimmiscible organic solvent. The organic extract can then de tested by application to filter paper, drying, and dipping or spraying with an alkaloid detecting reagent that giver a chromo genic response with alkaloids. If the latter method is not preferred, the organic solution can be re-extracted with dilute acid and the usual alkaloid precipitating reagents added to separate portions of this acid extract. Another method of removing impurities that are capable of giving false-positive tests from an initial aqueous acidic extract is to “salt out” these materials by the addition of powdered sodium chloride. An additional procedure for alkaloid detection could be based on the addition of alkali directly to the powdered plant sample, followed by extraction with an appropriate organic solvent. This extract could then be purified by partition as described above, or be tested directly. With respect to these general methods, certain anomalies have been reported in the literature which should be pointed out. There is no implication that these examples are frequently encountered in alkaloid screening: however, one should be aware that they do exist. Certain plants are known to contain labile non-basic constituents and may yield nitrogenous materials on extraction with ammoniacal solvents, while others contain alkaloids that are susceptible to modification by acidic reagents. That proteins, which may be present in aqueous or acidic aqueous plants extracts, can ppt. on the addition of heavy metal alkaloid precipitating reagents and thus yield false-positive tests, is well established. Such proteins can be removed by treatment of the extract with sodium chloride prior to the use of the heavy metal reagent, a procedure which usually salts out the protein. However, alkaloids such as alstonine may be quantitatively precipitated as hydrochloride under these conditions. In the treatment of a crude plant extract to remove impurities by the acid-base-organic solvent acid procedure, it is quite possible that 23

plants containing water-soluble alkaloid bases will go undetected. Quaternary bases, amine oxides, betaines, and choline would full into this category. Variability of results in alkaloid testing of plant material can, be induced by a number of factors such as age, climate habitat plant part tested season time of harvest chemical races of plants sensitivity of alkaloid type to reagents etc. A few examples regarding these factors should serve to point out their importance Geijera salicifolia was found by Webb to give consistently better alkaloid tests as the broad leaf form than narrow leaf form even when the 2 were growing side by side in the field. In certain groups of plants (i.e. composite) alkaloids often arc found only on or near the flower tops and in the Apocynaceae. Alkaloids generally tend to concentrate in the root or bark often to the exclusion of other parts of the plant thus the proper selection of plant parts for testing is quite important. To obtain equivalent results, quantitation of precipitates obtained with alkaloid reagent is not always possible, especially when comparing different genera or families. This is exemplified through knowledge that galbulimima baccata was found to be rated a ++++ in field tests and subsequent analysis resulted in a yeald of 0.01%-0.05% of 4 alkaloids. A++++ rating for Daphnandra aromatica was determined in the field and subscquent analysis in the laboratory yielded 6+% of crude alkaloids. Anlireha putaminosa loses 50% of its alkaloid decomposition rates have also been noted for A. tennuifolia randia rubiaceous plants. Silica gel drying of antirhea tennuifolia for 1 month resulted in material that gave a ++++ alkaloid test, whereas this same plant dried in the shade for 1month gave a negative alkaloid test. Acronychina baueri on the other hand gave strong alkaloid positive tests when 124year old herbarium specimens were evaluated Along similar lines , Raffauf and morris have reported that a plant sample identified as Nicoliana attenuata and estimated to be some 1300 years old gave positive alkaloid tests. Duboisia myoporoides yielded 3%of hyoscyaminne when harvested in October but when harvested in April of the same year 3% hyoscine was isolated. Example of alkaloid decomposition as a result of milling dried plant matcrial have also been cited. These examples should suffice to point out just a few of the problems encountered by the natural product investigator who is interested in the detection and isolation of biologically active alkaloids.

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SOME USEFUL ALKALOID PRECIPITATING REAGENTS NAME

COMPOSITION

Bouchardat

Iodine-Potassium-Iodide

Dragendroff

Bismuth potassium iodide

Ecolle

Sillicotungstic acid

Hanger

Picric acid

Kraut

Iodine-zinc chloriodide

Marme

Cadmium potassium iodide

Mayer

Potassium mercuric iodide

Platinum chloride

Chloroplatinic acid

Scheibler

Phosphotungustic acid

Sonneschein

Ammonium

hosphomolybdate Valser

Potassium mercuric iodide

Wagner

Iodine potassium iodide Bismuth antimony iodide Bromauric acid Bromoplatinic acid Bromothalic acid

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ALKALOID DETECTING REAGENTS For detecting alkaloids in phytochemical screening, two types of reagents are available, alkaloidal precipitants and spray or dip reagents. In table-1 20 precipitating reagents commonly used for the detection of alkaloids, whereas In table-2 present 25 reagents that were used in 45 recent phytochemical surveys for alkaloids. At least 2 reagents were used in 38 of the surveys, while 7 surveys depended solely on 1 reagent to establish the presence of alkaloids. Because of the variable sensitivities of these reagents and because of their nonspecificity for alkaloids many investigators utilize 4 or 5 reagents in their screening of plant extracts, and only samples yielding precipitates with all reagents are considered to contain alkaloids. Fulton has tabulated some 200 of these reagents and presents a great deal of information concerning their specilicity and sensitivity. A series of papers by munch a al is concerned with the effect of 17 different alkaloid detecting reagents on several classes of nitrogenous bases. Travell has studied the sensitivity of mayers and valsers reagents both solutions of potassium mercuric iodide and potassium iodide and the letter from mercuric iodide and potassium iodide. The reagent used by most investigators for phytochemical screening is essentionally the same formula that Mayer originally introduced in 1862. Several investigalors have demonstrated. How ever. That the original formula is perhaps the least sensitive for alkaloid detection in comparison with many proposed modifications. And travel has conclusively demonstrated the superiority of several common alkaloid precipitating reagents using 40 different chemical types. The reagents tested were mayers. Velsers wegners Bouchardats hagers Schreibers. Silicotangstic acid. Dregendorll”s Marme’s gold chloride.and sonnenschein’s. It was demonstrated in this study that the various reagents exhibit wide differences in sensitivity for structurally dissimilar alkaloids. None of the reagents would detect ephedrine at a concentration of 0.1% but wagner’s bouchardat’s. Dragendorff’s and scheibler’s each defected all of the other alkaloids at concentrations ranging from 0.001 to 0.1%.Hager’s marme’s and gold chloride reagents were by far least effective detecting reagent failing to react with 13 12 and 10 respectively of the 40 test alkaloids. All 3 of the Mayer’s formulations were inferior to Valser’s reagent with respect to sensitivity and specificity of alkaloid detection. It should be pointed out that the majority of these precipitating reagents must be used to defect 26

alkaloids only in acid solution. And further more. That a large number of naturally occurring mononitrogenous plant principles will react to give false-positive tests. These will be discussed subsequently,

ALKALOID DETECTING REAGENTS EMPLOYED IN SCREENING PROGRAMS REAGENT

SURVEYS USED

Mayer reagent

39

Silicotungustic acid reagent

23

Dragondroff’s drop reagent

19

Wagner’s reagent

11

Dragendroff’s spray reagent

10

Sonnenschein’s reagent

09

Hanger’s reagent

07

Bouchardat’s reagent

03

Phophotungustic acid

02

Valser’s reagent

01

Chloroplatinic acid reagent

01

Chlorauric acid reagent

01

Sodium tetraphenylboron reagent

01

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Investigators who prefer to use spot tests, or those who prefer to chromatograph concentrated plant extracts for the detection of alkaloids, have a variety of available reagents the most widely utilized, however, are modifications of the original dragendorff drop test reagent, which produce orange to red colors with most alkaloids. Although a number of modified formula have been proposed, each reported to have advantages over the others, the 2 most frequently utilized in phytochemical studies are the 1951 munier and macheboeuf and the these and rather modifications. A literature search has revisited the availability of at least 15 modifications of the original Dragendorff drop test reagent which produce orange to red colors with most alkaloids. Although a number of modified formulas have been proposed each reported to have advantages over the other the 2 most frequently utilized is phytochemical studies are the 1951. munier and macheboeuf and the these and rather modification. A literature search has revealed the availability of at least 15 modification of the dragendroff spray reagent .we are prompted to study the stability and sensitivity of one of these modified reagents since a number of published reports had commented on the need for their storage under refrigeration with concomitant protection from light. It was determined that prepared concentrates of the 1951 Munier machcbocuf dragendorff’s reagent required a storage period of about I week prior to its use in the preparation of the diluted reagent. Also the diluted spray reagent should be stored for a minimum of 1 week prior to its use for alkaloid detection in order to obtain maximum sensitivity. The reagent maintained its stability and sensitivity for at least 6 months and no special storage conditions were found necessary. Some alkaloid detecting reagent are available which on reaction with certain groups of alkaloids or with specific functional groups produce characteristic chromogenic value in screening work but only after alkaloids have been determined in the sample being evaluated. A selected list of general as well as specific chromogenic reagents has been prepared and is presented in table.

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False-Positive Alkaloid Reaction – Mechanism for the reaction between alkaloids and detecting reagents are dependent chiefly on the chemical character of the reagent. Fulton claddifies alkaloidal precipitates as Those which react with basic compounds to form insoluble salts; examples are silicotungstic phosphomolybdic and phosphotungstic acids. a) Those which react with alkaloids as loose complexes to form precipitates; examples are Wagner’s and bouchardat reagents. b) Those which react to form insoluble addition products through the alkaloid nitrogen ; examples are the complex heavy metal salt reagents Mayer’s valser’s marme’s and dragendorff’s arrd . c) Those, which react through the attraction of organic acids with basic alkaloids to form insoluble salts. An example of such a reagent would be Hager’s (picric acid). Obviously these are rather nonspecific reactions and a number of nonalkaloidal plant constituents should be expected. These false positive reactions are most liable to occur when testing an extract that has not been treated by at least one acid base organic solvent purification. The most frequent false positive reactions have been attributed to the presence of proteins which precipitate on the addition of heavy metal containing reagents. Included in this category are ptomaine’s. At least one textbook has indicated that amino acids will also precipitate with general alkaloid reagent. However, a study by Winek and Fitzgerald appears to disprove this allegation. Among other substance reported in the literature as the cause of false-positive alkaloid reaction are certain glycosides, and carbohydrate, betaine, choline, purines, methylated amines, tannins and ammonium salts. Recently, we were able to show that previous positive alkaloid tests reported for extracts of piper methysticum were due to the alfapyrones: kawain, dihydrokawain, methysticin, dihdromethysticin, and yagonine. This prompted an investigation of the mechanism by which nonalkaloidal compounds are able to elicit a positive reaction with an alkaloid detecting reagent, in this case the modified Dragendroff reagent. It was determined that any non-nitrogenous 29

organic compound having conjugated carbonyl or lactone functions would react in a manner typical of alkaloids. These minimum qualifications are quite prevalent in natural products and undoubtedly many false-reactions are promulgated through this mechanism. Fortunately, the majority of compound with these functionalities can be separated from the alkaloids by treatment of the extract with base, followed by extraction with organic solvent which, in turn, is extracted with diluted aqueous acid. Another interesting report that is by Bings and Locker, who in 1949isolated 3 compounds from Melicope ternate which give precipitates with the usual alkaloid reagent and crystalline salts with acids. How ever these compounds contained no nitrogen and proved to be the completely alkylated hydroxylflavones meliternatin meliternin and ternatin . It would appear that in the light of our work the falsepositive test for alkaloids was due to the presence of the conjugated carbonyl in each molecule rather than as stated by the authors the fact that each molecule was completely alkylated. Presumably other flavones would react similarly as would most of the cardenolides and bufadienolides. Householder and camp have recently pointed out that treatment of plant extracts with ammonium hydroxide and acetone can give rise to artifacts which give positive reactions with the standard qualitative alkaloid test reagents. These investigators were unable to identify the condensation products formed in this reaction but presented evidence to show that the rate of formation was affected by exposure to light and the atmosphere. A recent report by Russian works presented evidence for the isolation of an alkaloid named rosmaricine from rosmarinus offcinalis , This anomaly of on alkaloid from a number of the mint family prompted wankert and co-workers to investigate the validity of the Rassion work. They found that rosmaricine was indeed not present in the plant prior to the addition of ammonia {used by the Russian workers in their isolation experiments}. And that this “alkaloid” was undoubtedly formed as a result of the action of the base on the precursor caruosic acid. Other anomalous alkaloid reactions have been mentioned in the literature but as yet they cannot be explained. For example. Samolus repens extracts give a black color and precipitate with Dragendorff’s reagent. We have observed this phenomenon frequently in the field testing of fresh plant material and have assumed the reaction to be one of free iodine in the reagent combining with starch to give a typical blue black cooler. 30

Extracts from Plagianthus divaricatus have been reported to give a pink cooler. But no precipitate, with dragendorff’s reagent. Webb has pointed out that about 9% of species of species tested in the filed for alkaloid were found to be false positive reaction following subsequent detailed laboratory analysis. On the bases of experience resulting form test on the some 25000 plant species Douglas has estimated that not more than 5% of initial positive alkaline tests have been found to se done to nonalkaloidal entities. False- negative alkaloid reaction – if one considers the nonheterocyclic nitrogen bases as alkaloids it will be noted that the greatest majority of these fail to react with the usual alkaloid precipipatating reagent. Certain examples of this can be documented. Also unless certain precautions are observed in the test procedure quaternary alkaloids and amine oxides will not be detected. That is if an acidic plant extract is treaterd with base and extracted with an immiscible organic solvent both the aqueous basic layer and the organic layer must be tested for alkaloids. In most alkaloid screening procedures that have been reported the basic layer has been neglected. Argument for this omission have been based on the assumption that quaternary alkaloids or amine oxide would not be expected to occur in plants to the exclusion of tertiary bases which would be detected by this procedure. Raffauf points out that this may be an erroneous assumption. Alkaloid testing of herbarium specimens – the validity of alkaloid test on plant material derived from herbarium sheets is open the question. Often for many and varied reasons it is difficult to find certain plants in their negative habitat at the time of collection of indigenous flora and this alternative to collection has been used by several investigators to survey a broad distribution of plant task. An obvious disadvantage in the use of such material is that usually only leaves and branches are available and instances are known where in plants contain alkaloids in other organs but their leaves and stems are relatively alkaloid free. Also, herbarium material is often quite old and a number of examples can be cited correlating alkaloid decomposition as a function of time. On the other hand plants 1300 years old have been repoted still to give alkaloid positive test. It is common practice in some herbaria to treat specimens with formali could very well decompose many alkaloids and mercuricwith certain alkaloidal precipitants to from abnormal precipitates. With Mayer’s reagent this is evidence by a bright orange precipitate this is evidence by a bright orange 31

precipitate with yellow streaks which eventually become red, and with bochar dat’s reagent a pale purplish brown precipitate is observed. In the some herbaria it is common to mark specimen in a manner that treatment of this type can be easily ascertained while in other that this practice in not carried out. Cainetal have indicated that equivalent results were obtained in their studies with fresh plant material dried herbarium specimen. Field test for alkaloids in plants-- Investigator searching for new alkaloids bearing species in remote or distant areas of the world often find it difficult to return to make additional bulk plant collection for laboratory study. Therefore, simple field tests for alkaloids have been developed which are sufficiently reliable to distinguish alkaloids, thus enableing bulk collections of these species initially and eliminating the need for a return expedition. These field tests can be classified in the following manner. Organoleptic Evaluation- it has been suggested by wabb that at least some of his collections of species for laboratory examination were made on the basis of taste, in conjunction with some knowledge of the botanical characteristics of the sample being evaluated. That is he avoided tasting plants in such families as the Anacardiaceae, Euphorbiaceae, etc, but bitterncss in a group such as the lauraceae, particularly if a cryptocarya, would suggest alkaloids and saponins can be made on the basis of taste, but only after considerable experience. Also bitterness in the inner bark of such groups as evodia, acoronychia and melicop in conjunction with an observation of yellow pigmentation is suggestive of the presence of alkaloids. While judgment such as approach.

Spot tests using alkaloid test paper Kraft has development a simple device for alkaloid detection in fresh plant material a process which consist of impregnating filter paper with Dragendorff’s reagent fallowed by drying. A plant part is incised with a razor blade and a small amount of juice is applied to test paper which, if alkaloids are present will give the characteristic orange color indicative of a positive test. This method has been applied to the field testing of about 1200 species of plant by nikonov and ban’kovskii who found it to be acceptable with certain resertions. They found it unsuited for plants containing pigment in the sap which masked positive reaction and it was further determined that protoalkaloids such as ephedrine were not determined. An important point that must be 32

emphasized is that workers stress that typical color of an alkaloid positive reactive must be observed within 30 sec. from the time that the sample was applied to the paper for a test to be considered valid. We have used paper similar to this in our laboratory for the detection of alkaloids in organic solvent during chromo graphic separations and found that the after sample solvent had evaporated in order for the reaction to take place.

Spot tests on paper using liquid reagents The most extensive phytochemical survey for alkaloid in the plant kingdom being conducted by scientists from the nature product section smith kline & French laboratories Philadelphia. This program was initiated in 1954 but extensive alkaloid testing did not bening until 1958. briefly their approach consist of semiconductor collection of plants all part s of the world, with tests for alkaloid test for made in the field on all accessible parts of each species. Those found to be promising as a source of alkaloid are collected at the test site in sufficient quantity to enable laboratory extraction of the alkaloid for subsequent pharmacologic study. Extract from all plants show to contain alkaloids by this field test of screening for several types of pharmacologic activity. The field test for alkaloid used by this group has been described and is essentially the same as that utilized by nikonov and bankovskii with the exception of the special test paper. Instead plants sap obtained by making an incision of the approate plant part is applied to filter paper dried and a microdrop of specially prepared dragendorff’s regent is added. Positive tests are evaluated as previously described. All positive field tests are confirmed by means of a laboratory alkaloid detection procedure. Of 25000 species evaluated in this manner to date about 10% have been recorded as alkaloid positive. About 5% of the plants shown to contain alkaloids by the field test were not confirmed by the laboratory procedure.

33

Abisch and reichstein have also utilized this spot test technique for alkaloid detection in their study of plants of the apocynaceae A sclepiadaceae and periplocaceae. However their extracts were prepared from dry plant material.

Test Tube Spot Tests – Culvenor and Fitzgerald have described a simple kit that can be taken into the field for use in testing samples of plant material for alkaloids. About 2-4 gm of fresh plants part is ground in a small mortar with sand and sufficient chloroform to make slurry. Ammoniacal chloroform is added and the mixture stirred for 1min prior to filtration into a small test tube. Extraction of the alkaloids from the chloroform is accomplished by shaking the solution with 0.5ml of 2N sulfuric acid and separation of the acid layer by means of a medicine dropper. A few drop of this acid extract are then tested with either Mayer’s reagent orsilicotungstic acid to ascertain the presence of alkaloids. When samples were analyzed by both the field method and a laboratory procedure it was found that a number of weakly positive tests recorded through use of the laboratory test were found to be negative in the field test. The method of course fails to detect quaternary alkaloids and this appears to be its major draw back. Presumably many of the plantscollected for laboratory alkaloid testing by Webb, Amarsingham and Arthur were field analyzed in a similar manner to that describe above. However, their respective reports failed to point out any consistency with regard to this matter.

Alkaloid SurveysAlthough surveys for alkaloids, representing tests on more than 15,000 species of plants, have been published, the data that they present are often inconsistent because of variations in testing methodology. That is, some of the procedure will detect both quaternary and tertiary alkaloids, but the former group is omitted from most surveys reports. Certain procedures involve treatment of the alkaloid fraction to remove substances that often give rise to false-positive alkaloid reaction, whereas others do not include this extra step. Some methods are semiquantitative, while others lack this desirable feature. A survey of the most extensive 34

and more frequently reported methods allows them to be classified into 6 major categories. Perhaps the simplest method is that represented group by group A in which either an acidic or aqueous plant extract is prepared, with or without the use of heat, followed by filtration and the addition of one or more alkaloidal reagent to separate portions of the filtrate. Most investigatiors assess a rating of o, or +1 to +4 on the lack of, or degree of precipitation following use of the reagents. However, there is seldom any indication of the alkaloids equivalent of these ratings. This undoubtedly could present a problem to either a novice or one who is attempting to duplicate results in a different laboratory. On the other hand, a person experienced in alkaloid screening can usually assess this +1to +4 rating system by a rule of thumb.The major drawback of this method is that it results in the greatest number of false positive reaction. An inspection of the compounds presented in fig 2 which are representative of a great number of nonalkaloidal plant eonsttuents capable of giving false-positive alkaloid reactions, shows that for the most part they would. Be soluble either aqueous or acidic media. Also although this method world not differentiate between quaternary and tertiary alkaloids, nether would it fail to detect one or the other. Group Testing differs from group A only in that the filtrate is made basic and extracted With an organic solvent {usually chloroform or ether.} followed by extraction of the alkaloids from the organic solution with dilute aqueous acid. The usual alkoloidal precipitants are then added to separate portions of the acid extract, This method had the advantage over the group. A procedure of eliminating a great number of compounds from the final test extract that are capable of eliciting false-positive alkaloid reactions; however, any quaternary alkaloids present would also be eliminated. Simple modifications in this method would allow one to test for the latter group of alkaloids. Since water will extract a number of non alkaloidal constituents from plants and because there is a possibility of free alkaloid bases existing in the plant as such and these would be water insoluble most investigators utilize alcohol or alcohol water mixtures as a primary extraction medium. Group c test methods involve preparation of an alcohol extract followed by removal of solvent and the addition of dilute acid to dissolve any alkaloids. Some investigators test the resultant acid extract directly and stop at this point. The advantages and disadvantages for this type of testing are similar to those discussed for the group a methods. 35

Others will confirm initial positive reaction following a base organic solvent acid extraction. These are the group C-2 methods which are designed primarily to eliminate substances capable of eliciting false positive alkaloids reactions. The group C-1 and c-2 methods. Were designed and used most extensively by wall and co-workers but there are 2 important features that should be discusscd concerning these procedures first the test involves pre-cipitation of free alkaloid bases from the initial acid extract with NaOH rather than with NH,OH Thus. if a majority of the alkaloids in the sample were phenolic in character (highly improbadle) the phenolates formed on the addition of fixed alkali would be insoluble in the immiscible organic solvent used for the extraction of the basic alkaloid-containing solution. Subsequent extraction with dilute acid would then result in a solution free from phenolic alkaloids and would therefore not be representative of the true alkaloid content of the sample A second problem associated with this method was recognized by the workers themselves after screening the first 4000 accessions Because they were experiencing a lesser number of positive results than would be expected from statistical averages they increased the concentration of test solution so that l ml would be equivalent to 4.0 gm. Of dry plant material previous test results In the serics wcre rcported on solutions which represented only 0.2 gm of dry pample this of course made any ncgative alkaloid test rcsults rcpored in the fist 4000 acccssions open to qucstion Rccognizing this problem plants yiclding negative rcsults from the latter group if availablc wcre rctestcd and the results included in rcports on the final 2000 accessions the method does not include specific provisions for the detection of quaternary bases but as indicated previously modifications could be made so that this procedure would detect these compounds.

36

Screening for Heterosides (Glycosides) Heterosides arc organic compounds in which a hemiacetal linkage usually connects the anomeric carbon of a sugar (glycone) with an alcohol or phenolic hydroxyl of a second nonsugar molecule (aglycone) this type of linkage rise to the so-called o-heterosides (e.g.,salicin) the most common type of Heterosides found in plants If the anomeric carbon of the glycone is attached to an aglycone through sulfur the S-heterosides are formcd (e.g.,sinigrin) A third group are the N-hcterosides which involve attachment of the glycone to an amino group of an aglycone (e.g.vicine,crotnoside) Finally, the C-heterosides involve a carbon of carbon linkage of glycone and aglycone (e.g.aloin). As a general rule plant heterosides are easily hydrolyzed with dilute acids or appropriate enzymes the Chcterosides arc a notable ex-ception as they are resistant to the usual type of acid hydrolysis and require ferric chloride for this purpose. A number of different sugars are known to occur in plants in combination with an equally large number of diverse aglycones Paris has recently reviewed plant heterosides with particular reference to the types and distribution in plats. In most instances the biological activity of heterosides can be attributed to the aglycone moiety the glycone is mainly associated with the degree or modification of activity primarily induced by the aglycone. However the cardiac heterosides can be pointed out as a group that have no useful biological activity unless the heteroside is intact thus we have the economically important saponin heterosides and the medicinally useful anthraquinone flavonid cyanogenetic isothioyanate and cardiac groups. From a chemical point of view there are 3 parts of the heteroside molecule that can be used as a material of detecting this group of compounds in plant material. First the hemiacetal linkage between aglycone and glycol is usually not associated with biological activity, nor can it be associated with any specific aglycone. This part of the molecule does not appear attractive as a means of detecting plant heterosides. Because of the usual correlation of biological activity with the aglycone moiety of heterosides and because this part of molecule often has chemical properties amenable to ready detections, most investigator have used it as a means of screening plant material indirectly for heterosides. 37

Detection of Glycosides If, however, heterosides must be intact to exert their potential biological activity it would appear most fruitful to detect the hemiacetal linkage in plant extracts as an identifying feature of the presence of heterosides. Several investigator have proposed application of those to the screening of plants for hetersoidal attests to their complexity or inefficiency. Bourquelot proposed a method for detecting and identifying hetersoidal based on the determination of an “Enzymolytic index of reduction “obtain by measuring the optical reaction of a heteroside-containing plant extract before and after hydrolysiswith specific enzymes. Although the method has some value it is time consuming and requires large amounts of plant matcrial therclorc it would be difficult to adopt to a large-scale screening program Bliss and Ramstad devised a simple procedure that could be adapted for routine screening. It consists of (a) Separation of the heterosides in an extract by paper chromatography (b) hydrolysis of the heterosides on the chromatogram with proper enzymes (i.e.a-glucoidase-invertin-Bglucosidase-emulsin) and (c) Location of the reducing sugars formed on the chromatogram by means of an appropriate reagent spray. Tahis method appears to be least objectionable of many proposed. However it will detect only those heterosides for which the selected enzymes have a hydrolytic specificity Also optimal reaction conditions such as time temperature and pH would have to be determined for a large number of substrate heterosides to propose operating conditions that would allow detection of the greatest number of compounds. Janot et al and Paris have suggestcd chromatographic methods for detecting heterosides similar to the method of Biss and Ramstad but acid hydrolysis of the sample is induced to supplement the action of enzymes. Other methods have been proposed ,but ether they have not been applied successfully to plant sample or certain limiting factor make them of doubtful value for general screening. Knapp and Beal have proposed a method involve the selective extraction of heterosides from plant material using 80% ethanol oxidation of the free sugar in the extract to their corresponding carboxylic acids so that they will not be detected after hydrolysis of the heterosides hydrolysis of the heterosides in the extract using 0.15 N sulfuric acid and hart (100) and (d) detection of hydrolyzed glycones by means of paper chromatography. The major objection 38

to this procedure is that holosides, especially sucrose which is wide spread in plants, are detected thus the method is of decreased value. Abich and Reichstein have utilized a rather simple procedure which involves the preparation of an extract devoid of free sugars hydrolysis of the extract with the Killani acid mixture and testing of the hydrolysis products with Fehling’solution for evidence of reduction. These investigators have pointed out the nonspecificity of the test; however in a broad screening program false-positive reactions must be accepted especially in the presence of a completely acceptable and specific method of detection. It does not appear that adequate methodology has been developed to allow for an extensive screening of plants for hetrosides based on the approaches described above. As indicated previously, the majority of studies involving a search for hetrosides in plant material concerned with tests designed to detect specific aglycones. The more important of these will now be considered.

Isolation of triterpinoid glycosides This study reports the isolation and characterization of a new triterpenoid glycoside extracted from the bark of Terminalia arjuna. Theisolation of the organic compounds was done using simple chromatographic technique. Compound characterization using various spectroscopic technique identify the final isolated compound as Olean-3 ,22 -diol-12-en-28 Dglucopyrano -side-oic acid. The method of isolation is simple, cost effective andefficient. The preliminary bioactivity of the compound was also evaluated.

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Extraction and isolation of glycosides The sun-dried stem bark was crushed into fine powder. Pulverized bark part 2.5 kg was exhaustively extracted with ethanol (95%) at room temperature, 23 ± 2 oC, for 10 days. The ethanolic extract was filtered, distilled and concentrated to obtain the solid brownish residue (M.P = 158 oC). The yield was 7.1% w/w. The final weight was noted and stored. The residue was treated with water. The water soluble and insoluble portions were separately collected by filtration (G4 crucibles). Initial study in preparative TLC of the water-soluble part does not give any spot in the chromatogram and therefore, we did not take any further attempt to analyze the watersoluble part. The water insoluble alcohol extract was found to be partially soluble in different organic solvents like ethyl acetate, benzene, chloroform, carbon tetrachloride and methyl alcohol. So, it was dissolved in ethyl alcohol and allowed to stand for nearly 4 h then filtered and separated into ethyl alcohol soluble (A) and insoluble (B) parts.The ethyl alcohol soluble (A) portion was treated with equal volume of distilled water and ethanol mixture (1:1) and then treated with ethyl acetate in separating funnel, which separated into organic layer and the aqueous layers. The process was repeated for at least three times to ensure complete extraction From the organic layer taken separately, the ethyl acetate was distilled out by and it was further treated with dry petroleum benzene and the purity of the compound was tested using thin layer chromatographic system developed in a benzene and ethyl acetate (9:1)solvent system. Three different spots were obtained when the chromatogram was placed inside an Iodine chamber, indicating the presence of three different compounds. All the three compounds were separated and collected using preparative thin layer chromatography. However, we failed to get a quantitative yield of the materials and therefore, further analysis of the compounds was not undertaken in the present investigation.The aqueous layer obtained in the above process was treated with distilled water and filtered. In this case, the thin layer chromatography developed in benzene and ethyl acetate (9:1) solvent system does not yield any spot thus confirming absence of any compounds. The residue portion (B) was refluxed with petroleum benzene for 12 h using 40

reflux condenser and water bath. It was filtered and separated into two parts, residue (C) and filtrate (D). The filtrate (D) was subjected to preparative TLC and no spot was found, thus confirming absence of any compound. The residue (C) was again refluxed with dry benzenefor 12 h and filtered. No residue was obtained in this case. So, the benzene soluble portion was concentrated using a hot water bath to obtain a greenish-white colored residue. This wasfurther treated with petroleum benzene mixture (9:1) and recrystallized in benzene to give a white color compound (D), (M.P. = 160 - 162 oC), with quantitative yield.

Screening of Anthraquinones The largest groups of naturally occurring quinine substances are the anthraquinones. Although they have a widespread use as dyes, their chief medicinal value is dependent upon their cathartic action. They are restricted distribution in the plant kingdom and are found most frequently in members of the Rhamnaceae, Polygonaceae, Rubiaceae, Leguminosae and Liliaceae. As found in plant, they are usually carboxylated, methylated or hydroxylated derivatives of the anthracines, anthrone, anthranol, anthraquinone, or dianthrone. Hydroxylated anthracines often occur as hetrosides linked with various sugars through one of the hydroxyl group. Other types of anthracene hetrosides are represented as C-hetrosides in which the sugar and aglycone are linked by a carbon to carbon bond.

DETECTION OF ANTHRAQUINONES For the qualitative detection of anthraquinones in plant material, the Brontrager reaction, as modified by Kraus, appears to be simplest to perform in the application to phytochemical screening. The powdered sample (0.3gm) is boiled for a few minute with 0.5 N KOIT (10ml) to which is added 1 ml. of dilute hydrogen peroxide solution. After cooling the mixture is filtered and 5 ml. acidified with 10 ml. of benzene in a separator and the benzene layer takes on a yellow color . A 5-ml. sample of ammonium hydroxide and a positive reactive for the presence of an thraquinones is evidence by the formation of a red color in the alkaline layer. normally if c-glycosides are present in a sample 41

being evaluated for anthra-quinones they will not be detected by the usual Borntrager reaction as c-glycosides require special methods for cleaving the sugar from the aglycone this can be done with ferric chloride sodium dithionate or as described above with peroxide in an alkaline medium It has been shown that this method results in a mixture of products however this is not a disad vantage for a general screening test other simple and rapid spot tests which involve the direct addition of a reagent to the solid sample (powdered drug) have been described they should be useful in phytoehemical screening but to date have not been shown to be ap-pliable for this type of work phytoehemcial surveys for anthraquinones have been found only infrequently in the

Screening of Tannins Two groups of phenolic constituents, hydrolysable and condensed, comprise the tannins, substances which are important economically as agents for the tanning of leather and for certain medicinal purpose. More recently, evidence has been presented in support of their potential value as cytotoxic neoplastic agents.

Properties of tannins Hydrolysable tannins are yellow-brown amorphous substances which dissolve in hot water to form colloidal dispersions. They are astringent and have the ability to tan hide. Chemically speaking, they are esters which can be hydrolyzed by boiling with dilute acid to yield a phenolic compound, usually a derivative of gallic acid, and a sugar. These are often referred to as pyrogallol tannins. Condensed tannins are polymers of phenolic compounds related to the flavonoids and are similar in general properties to the hydrolysed tannins but are not very soluble in water and following treatment with boiling dilute acid red-brown insoluble polymers known as phlabaphenes or tannins-red are formed. Tannins are detected most simply in plant extract by the use of the so called gelatin-block test which has been utilized extensively in the phytochemical surveys. This test employs aqueous extract prepared from 80% ethanol extracted plant material. A sodium chloride solution is added to one portion of the 42

test extract, of 1% gelatine solution to a second portion, and the gelatine salt reagent to a third portion. Precipitation with the latter reagent or with both the gelatine and gelatine-salt reagents is indicative of the presence of tannins. If precipition is observed only with the salt solution a false-positive test is indicated. Positive test are confirmed by the addition of ferric chloride solution to the extract and should result in a blue, blue-black, green or blue-green color and precipitate. Hoch has applied some 33 different classical tannin detecting reagents to several tannin extract; however, the nonspecificity of many of these would render them impractical for use in general phytochemical screening work.

Extraction of tannins The process used to extract tannins is the hydrosolubilization Because this process operates with temperaturesaround 100°C, the extraction process motives ahydro cracking of sugar and others organic compounds with a darkening of the final product. We studied the supercritical extraction process as alternative procedure to obtain the natural raw material to leather tannage . In which supercritical carbon dioxide and polar ornon-polar co-solvents were used as solvents. The advantages of supercritical extraction process withregard to hydrosolubilization and solvent extraction are low extraction temperature, shortextraction time and absence organic solvent concentration in the extract. Another objective wasto compare the different extraction technology.

MATERIAL AND METHODS Natural Material The raw material, black acacia bark, was provided by EXTRATOS BRASIL. A part of bark was milled with cutting mill (TECNAL - Willye TE 650) with 2.0 mm average particle diameter. In the solvent extraction with Soxhlet apparatus, dry natural material was employed. The dryer used Pansera, M. R. et al. Brazilian Archives of Biology and Technology 996 was a BIOMATIC Equipment. The tannin was dried for 10 days at 36°C. 43

Solvent Extraction Process Samples (50 g of dark acacia bark) were extracted with 500 mL of solvent for 24 h. The extracted solution was concentrated on a SAVANT Lyofilizator.

Supercritical Extraction Process A Hewlett Packard 7680 extraction module was used to perform all the experiments. This system consisted of a nozzle/trap assembly that acted as a controllable variable restrictor, allowing an instantaneous depressurization of the supercriticalfluid as well as the decoupling of flow and pressure. The material to be extracted was loaded into a self-sealing extraction cell of 7.0 mL thickwalled stainless steel thimble. Supercritical fluid extracts were deposited in an internal trap rinsed off into a vial with 1 mL of hexane and methanol. Samples (0.4 g of bark) were extracted with supercritical carbon dioxide according to the described procedure, where different experiments to optimize the extraction conditions were done.The operation temperature and pressure rangestested were 40 to 80ºC and 150 to 200 bar,respectively. All other variables were kept constant: CO2 flow, 2.0 mL/min; and extractiontime, 30 minutes..

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Essential oils Essential oils (volatile oils) in addition to their value as flavoring agents and perfumes have been reported to have excellent antibacterial and antifungal properties. A few reports have been published which inclued an evaluation of plant samples for the presence of essential oils For maximum efficieney tests should be conducted on fresh material since most of the volatile constituents of plants are lost during drying. In most instances the methods that have been employed since they of necessity had been conducted in the field as organoleptie examina-tions. More elaborate laboratory examinations have involved steam distillation followed by measurement of the water immiscible oil and in some eases followed by the application of chemical tests for terpenes Arthur studied more than 700 species in North Borneo and Hong Kong and simply chopped a small amount of fresh plant with a razor introduced the into a test tube added hot water and boiled the mixture Any characteristic odor of essential oils was then recorded. Kohlmunzer evaluated some 59 species of plants from genera known to have previously yielded economically important essential oils (salvia Lavandula Mentha Rosmarinus Thymus etc). Following steam distillation and subsequent measurement of the separation oil, chemical tests for cineol were applied on the other hand Betts has devised a method employing thin layer chromatography for the evaluation of petroleum ether extracts from umbelliferous fruits in which essential oils are universally soluble. Fluorescein-treatad plates of his extracts were first viewed under ultraviolet light to note the presence of dark quenching spots against a bright yellow background Unsaturated compounds were then detected as spots by exposure of the plates to bromine vapor which converted the fluorescein to eosin and subsequent ultraviolet examination then indicated unsaturated compounds against a dull background plates were then sprayed with 2,4-dinitrophenylhydrazine which revealed ketones and aldehydes as orange spots. A simple microcohobation still for the estimation of quantities of essential oil ranging from 250 al. in small (0.4 Gm.) samples of plant material has been shown to screening large numbers of plant samples for essential oils on a quantitative basis. At least this would be some improvement over current organoleptie methodology.. Extraction: Hydro distillation method was used for the extraction of the oil. The macerated plants were weighed and packed into the 45

round bottom flask. This was mixed with lots of water as the extracting solvent. The distillates were then transferred into a sample bottle. Antibacterial assay: Pour plate method was used for the assay . Nutrient agar was prepared by dissolving the appropriate quantity in deionised water, homogenized and sterilized. The organism was introduced into the sterile plate under aseptic conditions. 20ml of the prepared nutrient was also poured into the petri dish, swirled together and allowed to solidify. After solidifying, cork borer(9mm) was used to bore two wells in the medium or culture and the oil poured into the bored well to notice if there would be any zone of inhibition or not. A positive control with Streptomycin was used. Micro-organisms assay: The slants of nine organisms, six bacteria and three fungi were collected from the laboratory stock of the Department of Medical Microbiology, University College Hospital UCH, Ibadan, Oyo state. The bacterial are Klebsiella pneumoniae, Bacillus megaterium, Bacillus subtilis, Proteus mirabilis, Pseudomonas aeruginosa and Eschrichia coli and the fungi are Aspergillus favus, Trichoderma spp. And Aspergillus niger.

SCREENING OF Flavonoids 46

Definition & Chemistry The term favonoid refers to a class of plant secondary metabolites based around a phenyl benzopyrone structure. Flavonoids are most commonly known for their antioxidant activity. Flavonoids are also commonly referred to as bioflavonoids in the media –these terms are equivalent and interchangeable since all Flavonoids are biological in origin. The Flavonoid synthetic pathway begins with a product of glycolysis phosphoenolpyruvate entering into the shikimate pathway toyield phenylalanine. Phenylalanine is the starting material of the phenylalanine metabolic pathway from which 4coumaryl- CoA is produced. This can be Combined with malonyl – CoA to yield the true backbone of Flavonoids a group of compounds called chalcones. Ring-closure of these compounds results in the familiar form of Flavonoids a three ringed phenolic structure (polyphenols). Flavonoids have been referred to as “nature’s biological response modifiers” because of strong experimental evidence of their inherent ability to modify the body’s reaction to allergens viruses and carcinogens. They show anti-allergic anti-inflammatory anti- microbial and anti- cancer protectingagainst oxidative and free radical damage.

Isolation of Flavonoids It is estimated that about 8%of all carbon photosynthesized by plant is converted into flavonoid closely related compound most tanning are flavonoid. Flavonoid thus constitute one of the largely occurring natural compound. In plant flavonoid aglycone occur in variety of the structural form all contains 15carbon atom in main basic nucleus and these are arranged in a C-C-C configuration. That is two aromatic ring linked by 3corbon unit which may not form a third ring. The flavonoid varied are all related by common biosynthetic pathway which incorporate preculor form both the shikmate and acetate mevalonate pathway. These compounds occur in green plant with the exception of algae and horn weeds. They are virtually in all plant parts including leaves, wood, bark, pollen nector flowers brarriers and seeds in the few recoded cases of the favonoids being found in animals. The distribution of flavonoids is found in the largest way in the higher plant Angiosperm. Important feature of the distribution 47

of Flavonoids in plants is the strong tendancy taxonomically related plant to produce similar type Flavonoids.

ISOLATION-SOLUBILITY CHARACTERISTICS Flavonoid aglycone polyphenols and as such passes the property of phenotic I. e. they are slightly acidic and will thus dissolved in alkali. However it is left in alkali in the presence of oxygen many flavonoid will degrade flavonoid possess NO of unsubstituited –OH sps or sugar or polar compound. These are generally moderator dissolved in polar solvent such as methanol ethanol acetone dimethyl sulfoxamide dimethly furomide water etc presence of unattached sugar tends to render the flavonoid more H2O soluble while polar aglycones such as isoflavone flavonone and slightly methoxylated flavone and flavonones tends to be more soluble in solvent such as ether and propanol.

CONCLUSION 48

Thus the screening of the phytochemical constituent was studied screening can be used in research & development for specialization in field of pharmacy.

References 49

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12. Farsworth N R, Henry L K Svoboda G H, Blomster R N ,Yates M J and Euler K l. Biological and phytochemical evaluation of plants. I. biological test procedures and results from two hundred accessions. Lloydia (1966)29:01-122 13.Risk A M. Constituens of plants growing in Qatar. I a chemical survey of sixty plants. Fitoterapia(1982)52:35-44 14.Saheli Surmaghi M H, Aynehchi Y Amin GH and Mahmoodi Z. Survey of Iranian plants for saponins, alkaloids, flavonoids and trannis. 4. Daru (1992)2;281-291 15.Somolenski S J, Silinis H and Farnswoth N R. Alkaloid screening. I.Lioydia(1972)35:1-34 16.Somolenski S J, Silinis H and Farnswoth N R. Alkaloid screening. V. Lioydia(1974)37:506-536 17.Kapoor L D Singh A kapoor S L and Quimly M D. Falsenegative saponins test results induced by the response of tannins. Lloydia(1969)32:279-304 18 Segeman A B Farnsworth N R. Biological and phytochemical screening of plants.4. A new rapid procedure for the simultanoius determination of saponins and tannins. 19.Harborne J.B. 1973.Phtochemical Methods. Chapman & Hall London. 20. Harborne J.B. and B. L. Turner, 1984 Plant Chemosystematics. Academic Press, London and New York. 21. Heywood V .H. 1976. Plants taxonomy. The institute of Biology’s Studies in Biology No. 5.2 edition. 22. Willisms A. H. 1966.Dihydrochalcones.In T. Swain (ed.) Comparative Phytochemistry. Pp:297-307. Academic Press London, New York.

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