Final approved nature cures book draft Sept 7th 2022

other cellular components in the extracted DNA can consid- erably hasten the process of degradation. With increasing fragmentation of DNA template, its utility for providing useful information decreases progressively. Studies suggest that purified DNA dissolved in buffer, stores well up to 1-2 years at 40C, 4-7 years at -18 0 C and more than 4 years when stored at - 800 C. Its use in conservation is limited as whole plants cannot be reconstituted from DNA but the genetic material can be introduced to other genotypes for plant breeding and enhancement purposes. Its potential remains promising and should still be investigated as DNA is a very stable form in which genetic information can be stored (Andersson M. 2004). There are a number of areas in which DNA banks could make an impact in the near future. Most promising possibilities in this context are: Germplasm characterization and management: De- tailed characterization of germplasm using a combination of phenotypic and molecular markers improves genebank man- agement in several ways. It allows 1) detection of gaps in collections, identification of du- plicates and redundancy, 2) provides valuable knowledge about molecular diver- sity, genetic and evolutionary relationships, and 3) allows identification of unique genotypes of special importance to gene banks and breeders 51

Marker-assisted selection: An important new role for gene banks having DNA samples of germplasms is the appli- cation of molecular techniques to identify genes controlling specific traits in collections of cultivated species and wild relatives DNA barcoding: Global efforts are underway to produce DNA barcodes of all the plant species on earth. DNA banks would greatly facilitate such efforts by providing the required species DNA and thus avoiding the need for undertaking ex- pensive and time- consuming collection trips of depleting ra- re herbarium specimens. Exchange of genetic resources: It will be a lot easier to exchange genetic resources as DNA samples, rather than seed or vegetative propagules. Transboundary movement of seed and other planting material requires time consuming in- spection and certification for freedom from pests and diseas- es. Exchanging DNA samples, on the other hand, avoids the need for time consuming and costly certification procedures. 3.3.4. Pollen Banks Pollen preservation may be useful for base collections of species that do not produce orthodox seeds. It requires little space but some cytoplasmic genes would be lost. Like seeds, pollen can be divided into desiccation tolerant and intolerant. However, information about storage characteristics of pollen from wild species is fragmentary, existing mainly for some crop relatives and for medicinal and forest species. Desicca- tion-tolerant pollens are best stored at moisture levels of ≤ 10%, obtained by either air-drying or by equilibration at known RH. Greater longevities occur at lower temperatures, with more than 2 years often feasible with -18 °C (0 °F) stor- age. Cryogenic storage at -80 to -196 °C (-112 to -320 °F) 52

greatly increases longevity. Storage in a vacuum or with a N2 atmosphere also enhances longevity. Storage of desiccation- sensitive pollen is more problematic, but some can be desic- cated to 10 to 15% and preserved. 3.3.5. In vitro Storage Methods Although field gene banks provide easy access to con- served material for use, they run the risk of destruction by natural calamities, pests and diseases. For this reason, safety duplicates of the living collections are established using al- ternate strategies of conservation and it is in this area that bi- otechnology contributed significantly by providing comple- mentary in vitro conservation options through tissue culture techniques. In vitro conservation also offers other distinct advantages. For example, the material can be maintained in a pathogen-tested state, thereby facilitating safer distribution. Further, the cultures are not subjected to environmental dis- turbances (Withers and Engelmann, 1997). Several in vitro techniques have been developed for stor- age of vegetatively propagated and recalcitrant seed produc- ing species. In general, they fall under two categories: (i) slow growth procedures, where germplasm acces- sions are kept as sterile plant tissues or plantlets on nutrient gels provide short- and medium-term storage options. Slow growth procedures allow clonal plant material to be- held for 1 -15 years under tissue culture conditions with peri- odic sub-culturing, depending on species. There are several methods by which slow growth can be maintained in most cases, a low temperature often in combination with low light intensity or even darkness is used to limit growth. Tempera- tures in the range of 0-5ºC are employed with cold tolerant 53

species, but for tropical species which are generally sensitive to cold, temperatures between 15º and 20ºC are used. It is al- so possible to limit growth by modifying the culture medium, mainly by reducing the sugar and/or mineral elements con- centration and reduction of oxygen level available to cultures by covering explants with a layer of liquid medium or miner- al oil (Withers and Engelmann, 1997). (ii) Cryopreservation is the only available method for long-term conservation of vegetatively propagated plant germplasm. It involves storage of plant material at ultra-low temperatures in liquid nitrogen (-196°C). At this temperature, cell division and metabolic activities remain suspended and the material can be stored without changes for long periods of time. Cryogenic preservation of vegetative material is an- other mode of ex-situ conservation and it holds promise, es- pecially for base collections. The choice of material includes cells, protoplasts, shoot apices, somatic embryos, seed or ex- cised zygotic embryos. Cryopreservation is advantageous because it requires lim- ited space, protects material from contamination, involves very little maintenance and is considered to be a cost - effec- tive option. The techniques for cryopreservation currently in use are quite varied and include the older classical techniques based on freeze-induced dehydration of cells as well as new- er techniques based on vitrification (Engelmann, 2000). Engelmann described seven vitrification-based procedures in use for cryopreservation: (1) encapsulation-dehydration, (2) vitrification, (3) encapsulation-vitrification, (4) desicca- tion, (5) pregrowth, (6) pregrowth-desiccation, and (7) drop- let freezing. With the advent of these new cryogenic proce- dures, especially vitrification, encapsulation-vitrification and 54

encapsulation dehydration, the number of species cryo- preserved has increased significantly in recent years. 4. DIFFERENT INITIATIVES FOR EX SITU CONSERVATION: In India, Department of Biotechnology has sponsored network of four National Gene Banks for Medicinal and Ar- omatic Plants at 1. the Central Institute of Medicinal and Ar- omatic Plants (CIMAP), Lucknow; 2. Tropical Botanic Gar- den & Research Institute (TBGRI), Thiruvananthapuram; 3. National Bureau of Plant Genetic Resources (NBPGR), New Delhi and 4. Indian Institute of Integrative Medicine (former- ly known as Regional Research Laboratory), Jammu. Each gene bank is equipped with the facilities of a field bank, seed bank, in vitro bank and cryobank. While the TBGRI bank concentrates its activity on bioge- ographic sub-regions of the peninsular India, CIMAP and NBPGR are focusing on the northern regions. The fourth gene bank set-up at Indian Institute of Integrative Medicine (formerly known as Regional Research Laboratory), Jammu, has been developed to give special attention to threatened medicinal and aromatic plants of the North Western Himala- yas. IIIM, Jammu has initiated studies on ex situ conserva- tion and bioprospecting of biomolecules for sustainable utili- zation of medicinal plant biodiversity of Himalayan region. Initiative taken are- 4.1 Development of Repositories: • Field gene banks have been established at four differ- ent locations in Jammu and Kashmir at Yarikha (2500m), Bonnera (2000m), Srinagar (1700m) and Jammu (300m). A live plant repository of 243 medic- inal and aromatic plant species is maintained in these four field gene banks. 55

• Medium term conservation facility for seeds. A seed bank containing 108 plant species. • In vitro tissue of 40 medicinal and aromatic plants in different morphological forms. • A DNA repository containing DNA of 32 HMAP’s. Representation of MAPs conserved in different Gene Banks 120 100 80 60 40 20 Bonnera Srinaga Yarikha Jammu Seed Gene In Vitro DNA Bank repository 56

Table 1. Seeds of the Plant Species Stored At Low Temperature in Seed Bank Plant species Plant species Plant species Abelmoschus moschatus Datura metel Picrorhiza kurrooa Abutilon indicum Datura stramomium Pinus gerardiana Acacia Senegal Dodonaea viscose Pistacia vera. Achillea millifolium Dracocephalum heterophylum Pleurospermum candollei Achranthus aspera Echinacea angustifolia Podophyllum hexandrum (03) Adhatoda vasica Echinacea purpurea Polygonum rumicifolium Albizia lebbek Gloriosa superba (02) Prangos pabularia Ammi majus Grewia tanax Psorlea cordifolia Anacyclis pyrethrum (03) Hedychium spicatum Pueraria tuberosa Andrographis paniculata Heracleum candicans Quercus balooriss Angelica archangelica Heracleum pinnatum Ranunculus sclareatus Angelica glauca Hibiscus abelmoschus Rauvolfia canescens Argyrolobium roseum (10) Hippophae rhamnoides (06) Rauvolfia serpentina (04) Aristolochia indica Hordeum vulgare Rheum emodi Artemisia absinthium Hyocymus niger Rheum moorcroftianum (02) Artemisia annua Hypericum perforatum (05) Rhododendron campanulatum Artemisia dracunculus (05) Indigofera tinctoria Robina pseudoacacia Artemisia lacinata Iris kumaonesis Rosa webbiana Artemisia macrocephala Jatropha curcus (07) Salvia sclarea 57

Artemisia pallens Jurinea macrocephala Sapindus mukorossi Artemisia sieversiana Lilium thomsoni Saussurea lappa Arctium lappa (02) Lowsonia alba Saussurea simpsoniana Atropa belladonna Matricaria chamomilla Selinium tenuifolium Boswellia serrata Mecanopsis aculeate (02) Selinum candolli (02) Bunium pericum Mimosa pudica Silybum marianum (10) Bupleurum spp. Moringa olifera Stevia rebaudianan Capparis spinosa Mucuna pruriens Swertia chirata Carum carvi Nardostachys jatamansi Tanacetum gracile Cassia fistula Ocimum canum(03) Tanacetum longifolium Celastrus paniculatus (15) Ocimum clocimum (07) Tinospora cordifolia(02) Chaerophyllum villosum Oenanthe javanica Tribulus terrestris Clitoria turnatoa Oreganum vulgare (03) Verbascum thapsus Colebrookia oppositifolia Papaver somnifera Vetiveria zizinoides Crotolaria spp. Peganum harmala Withania somnifera (30) Cuphea latifolia Phyllanthus niruri Zanthoxylum alatum Cuphea maritime Physoclaina praelta (02) 58

Table 2: DNA OF THE PLANT SPECIES STORED IN DNA No. of Plant species Place of collection accessions 1 Acantholimon sp. Leh 4 Aconitum heterophyllum Kashmir 2 Acorus calamus Jammu field Gene Bank 3 Alpinia galanga Jammu field Gene Bank 2 Andrographis paniculata Jammu field Gene Bank 1 Arnebia benthamii Kashmir 1 Arnebia euchroma Kargil, Leh 2 Artemisia absinthium Leh 2 Artemisia dracunculus Leh / Lahaul 1 Artemisia laciniata Kargil, Leh 3 Bistorta sp. Kargil, Leh 1 Centella asiatica Jammu field Gene Bank 1 Cicer microphyllum Zanskar,Leh 1 Curcuma amada Jammu field Gene 2 Dactylorhiza hatagirea Leh 1 Delphinium ajacis Srinagar 2 Echinacea angustifolia Kashmir 1 Echinacea purpurea Kashmir 1 Heracleum candicans Kashmir 1 Hypericum perforatum Kashmir 1 Jatropha curcas Kashmir 59

Mentha longifolia Kashmir 1 Perovskia spp. Leh 1 Physochlaina praelata Leh 2 Picrorhiza kurrooa Kashmir 1 Podophyllum hexandrum Kashmir 2 Rheum australe Darjeeling NE 1 Swertia bimaculata GH, RRL Jammu 2 Swertia chirata Jammu field Gene Bank 1 Swertia chordata Jammu field Gene Bank 3 Tinospora cordifolia Jammu field Gene Bank 1 Withania somnifera Jammu field Gene Bank 5 60

Fig.1 61

4.2. In vitro conservation activities • Establishment of in vitro cultures repository of High altitude Himalayan Medicinal plants. • Development of high throughput regeneration proce- dures • Ex situ conservation through limited growth. • Bioresource bioprospecting 4.2.1. Establishment of in vitro cultures repository of High altitude Himalayan Medicinal plants Live plant material of rare, endangered or overexploited Himalayan species was collected from different altitudinal locations in North West Himalayas. Passport data regarding collection sites have been recorded as per standard guide- lines. In vitro axenic cultures have been established and pre- sent in vitro repository at IIIM, Jammu, holds 55 accessions of 40 medicinal plant species in various morphological forms, preferably as shoot cultures (Fig.2). Details of the cur- rent status of Himalayan medicinal and aromatic plants are given in Table 3. 62

Fig. 2 It is in Vitro cultures of various medicinal plants. 63

Table 3. In vitro Banking: Current Holdings Plant Species Morphogenetic Level of Differentiation Callus Shoot culture Plantlets Aconitum heterophyl- X X lum Acorus calamus XX Allium sativum X X Allium wallichi X XX Anglica archanglica X Artemesia annua XX Artemisa dracunulus X Artemisa moocroftiana X Artemisa vulgaris XX Atropa acuminata X XX Bunium persicum X X Crocus sativus XX 64

Dioscorea composita XX XX Dioscorea deltoidea X XX XX Drymaria cordata XX Echinecea purpurea XX X Hedychium spicatum XX XX Hyoscyamus muticus X XX Hypericum perforatum X X XX Picrorhiza kurroa XX Potentilla fulgens Rheum austriale X Rosmarinus officinalis Rubia cordifolia X Stevia rebaudiana. X Swertia chirata Valariana wallichii X 65

4.4.4. Development of high throughput regeneration pro- cedures Regeneration and successful propagation of genetically stable seedlings from cultures are prerequisites for any in vitro conservation effort. There are also a number of other important crop species that are sterile or do not easily pro- duce seeds, or seed is highly heterozygous and clonal propa- gation is preferred to conserve elite genotypes. Complete mi- cropropagation procedures have been developed for 25 high altitude medicinal and aromatic plant species given in Table 4. Some critically endangered/economically important ones are Swertia chirata, Picrorhiza kurroa, Crocus sativus, Atro- pa acuminate, Hedychium spicatum Artemisia spp., Hyperi- cum perforatum, Valariana wallichii Rheum austriale and Potentilla fulgens. (Fig 3). Procedure for progressing ad- vanced tissue culture research such as somatic embryogene- sis for Aconitum heterophullum, Bunium persicum has been standardized as a precursor to artificial seed production and microcorm formation for large-scale propagation. Fig. 3 66

4.4.5. Ex situ conservation through limited growth Standardization of Tissue Culture procedures for ex situ conservation through limited growth, culture of High- Altitude Medicinal Plants and slow growth /restricted growth on minimal nutrient media having growth retarding substanc- es with low temperature (5°C-10°C) incubation have been developed for. About 30 genera are presently preserved at various mor- phogenetic levels such as callus, somatic embryogenic cul- tures, multiple shoot cultures root cultures and suspension cultures Slow growth conditions are most suitable for preser- vation of plant species, the material requires being readily available for regeneration, multiplication, distribution and bioprospection. By limiting the number of subculture signifi- cant saving in labour input and minimizing risk of metabolic suppression of growth can be achieved with reduced light conditions and media components concentration. By adopting standardized procedure High Altitude Medic- inal Plants could be successful conserved in vitro for duration ranged between 1.5 months to 15 months depending on the species. Developed Excised Root Cultures and encapsulation as an approach for conservation and propagation of Atropa acuminata, Swertia chirata, Celastrus paniculatus, Artemisia dracanculus, Valeriana wallichi and Withania somnifera. Es- tablishment of axenic root cultures, their differentiation to complete plantlet formation and development of medium term conservation procedure has been standardized for Picrorhiza kurroa and Swertia chirayita. 67

Fig. 4 4.4.6 Bioresource bioprospecting In recent years there has been dramatic progress in the use of in vitro system to synthesize substances of pharmaceutical and nutritional importance. Depending upon the production capabilities of the particular cells conditions can be opti- mized for the production of specific cells or organ to synthe- size desired compound. These may take the form of cells, callus, somatic embryos or organs such as roots. All will be special in terms of their synthetic capacity. In vitro organ culture as alternative source of bio- mass/biomolecule production for critically endangered/elite strains has been worked out for Swertia chirayita. Conditions have been optimized for shoot biomass and amarogentin and amaroswerin production in 10 L air lift fermentor with 3 L working volume. Table 5 lists plant species where studies have been conducted on production of secondary metabolites from different morphotypes of plant cell cultures. 68

Fig. 5 Table 4. Details of in vitro Establishment, Micropropagation and Conser- vation of different High Altitude Himalayan Plant Species of Medicinal value Species Explant Multiplication -3 Rooting Sub-culture Hardening BM+ PGR frequency at Status BM + PGR (mg (mg dm-3) dm) Standard Storage Condition Aconitum het- Seedlings MS + BAP 0.5 - 2 Months Standardization erophyllum under process Acorus cala- Shoot Bud MS + IBA 0.1 + MS + IBA 2 months Establishment mus BAP 1.0 0.1 + BAP in Field 1.0 Allium wallichi Bulbil MS + IBA1.0 MS + 1.0 8 months Earthen Pots + 1.0 BAP IBA + (GH) 1.0 BAP 2.0 69

Angelica glau- Shoot Trip MS + IBA 1.0 + - 4 months Standardization ca Kn 1.0 5 months under process Unripe B5 + IBA Establishment Argyrolobium Embryos MS + Kn 1.0 + 0.5 in Field roseum NAA 1.0 Standardization Artemisia Shoot Tip MS + IAA1.0 + MS + 1.0 1 month under process moorcroftiana IBA1.0 IAA Earthen Pots Artemisia Shoot Tip/ MS + Kn 4.0 + MS + 6 months (GH) dracanulus Node IAA0.5 IBA2.0 Establishment Artemisia vul- Shoot Tip/ MS + BAP0.5 + MS + 1.0 5 months in Field garis Node IAA1.0 IAA Establishment Atropa acu- Shoot Tip/ MS + BAP 1.0 + RT + 1.0 6 months in Field minata Node IBA1.0 IBA Earthen Pots Bunium persi- Leaf MS + BAP 1.0 + MS 5 months (GH) cum segment NAA 1.0 + GA 3.0 Microtu- Earthen Pots Celastrus blers MS+NAA 0.95 WPM + 5 months (GH) paniculatus 1.0 IBA + Seed Em- 0.1% AC Earthen Pots bryo (GH) Crocus sativus Bulblets MS + BAP 1.0 + MS + BAP 5 months Establishment NAA 1.0 1.0 NAA in Field 1.0 + AC RT + BAP 1.0 Eartthen pots 2% (GH) Dioscorea Shoot Tip RT + BA+ 5 months deltoidea NAA1 + Drymaria cor- Shoot MS + IAA 1.0 MS 0.5 6 months data Tip/ BAP + 1.0 Nodes IAA Hedychium Rhizome MS + IBA 1.0 + MS + IBA 12 Months Established in spicatum segments BAP 1.0 1.0 + BAP Pots (GH) 1.0 70

Hypericum Shoot MS + IAA 1.0 MS + IAA1.0 6 Months Establishment in perforatum Tips Field Onosma Kn Eartthen pots echoides RT+ 2.0+ (GH) Shoot Tip RT + Kn 2.0 NAA 8 Months Picrorhiza Leaf MS + BAP 1.0 2.0 kurroa Segment Earthen Pots Shoot (GH) Tip MS + IBA1.0 5 Months Polygonum Shoot MS + BAP 1.0 MS + Eartthen pots amplexicaule Tip BAP1.0 + (GH) IAA 6 Months 1.0 Polygonum Shoot MS + BAP1.0 + Eartthen pots runicinatum Tip (GH) IAA1.0 MS + IAA1.0 6 Months Potentilla Shoot RT + Kn 1.0 RT + Kn 1.0 6 Months Establishment in fulgens Tip Field Rauwolfia Nodal M IAA/IB Establishment in serpentina Segments MS + BAP 1.0 + IAA S + A Field 2.0 8 Months 2.0 Rheum australe MS + NAA 1.0 Standardization Seedling MS + NAA 1.0 + BAP + under 1.0 BAP 3 Months Progress 1.0 Shoot Expt in Standardization Tip under Rheum emodi MS + IBA 1.0 BAP 1.0 Progress 2 Months Progress 71

Swertia AxillaryBud MS + BAP 0.5 MS + IAA1.0 2 Months Standardization angustifolia Shoot under Meristem Progress MS + BAP 0.5 MS + 1.0 IBA AxillaryBud MS + BAP0.5 + Earthen Swertia chiryata Shoot IAA1.1 Pots(GH) NAA1. 5 Months Meristem +0 Tancetum Shoot MS + 1.0 BAP + 1.0 MS + 1.0 Standardization longifolium Tip IBA IBA 2 Months under Progress Shoot Expt in Sassuria sacra Tip MS + IBA 1.0 + Kn 1.0 Progress -- - GH=Green House 72

Table 5. Bioresourcing Plant Tissue Cultures As An Alternative Means For Production PLANT SPECIES PRODUCT TISSUE TYPE Ammi Coumarins Callus & Shoot cultures majus Ammi visnaga Coumarins Callus, shoot & root cultures Argyrolobium roseum Iridoid glycosides Shoot cultures Atropa acuminata Tropane alkaloids Shoot & Root cultures Bacopa monnieri Triterpenoids Shoot cultures Cassia Anthraquinones Callus, shoot & root cultures fistula Datura inoxia Tropane alkaloids Callus & shoot cultures Dioscorea deltoidea Diosgenin Callus, shoot & root cultures Hyoscyamus muticus Tropane alkaloids Callus, Suspension & Shoot Cultures Hypericum perforatum Hypericin & Hyperforin Callus & Shoot Cultures Onasoma echoides Naphthoquinones Callus & root cultures Picrorhiza kurroa Iridoid glycosides Shhot & root Cultures Withania somnifera Withanoloids Callus, shoot & root cultures References: Appendix II – page 189 Contact: Dr. Sushma Koul [email protected] 73

Hippophae Rhmnoides from Leh (Laddakh), Science Citation: Surinder Kitchlu 74

Chapter 3 Phytochemicals as Immunomodulatory agents Author Dr. Anpurna Koul Introduction: Phytochemicals are the latest weapon in the fight against diseases. Phytochemicals are the next level in achieving overall good health. They are not vitamins, minerals, fiber, amino acids or enzymes, yet they are the latest weapons in the fight against disease. They are the natural chemical com- pounds found in all plants; they protect against diseases and promote health, and there are thousands of them. Phytochem- ical simply means foods with the ingredients thought to pre- vent disease. There is no doubt that food is what provides building blocks for our body’s healing mechanism. But there is no more food than just the vitamins and minerals that are essential to build and maintain optimum health. Phytochemi- cals, Phytonutrients, Phyto-foods, Functional foods, are the next level in achieving overall good health. Nutritionally high-powered foods aren’t new. But science’s added knowledge about the disease-preventing components these foods contain is ―new. The National Cancer Institute has launched a multimillion-dollar study of phytochemicals. Sci- entists have already discovered 30 phytochemicals that might help prevent cancer. Phytochemicals: Phytochemicals are chemical substances found in plants that can help prevent diseases and cell damage. Over one thousand phytonutrients have already been identified. Phyto- nutrients can play many roles in keeping the body healthy. These are natural chemicals in plants which are not nutritious but that protect and help to prevent from diseases. Plants will produce phytochemicals as a protection device. There are 75

over a thousand of these substances known already, and more are being discovered all the time. Some of these substances are becoming well known, such as the lycopene that make tomatoes so good for you and the isoflavones that are found in soy. Flavanoids, which are found in fruits, are another of the well-known phytonutrients. These substances are not considered essential to life, but they can really help in staying healthy and prevent from a number of different diseases. Phytonutrients work in a number of different ways, de- pending on the specific substance. Many are antioxidants, and help to protect against cancer risks and other cell damag- es. This includes the carotenoids which is found in carrots and many fruits, the polyphenols which are abundant in grapes and certain types of teas, the allyl sulphides contained in garlic, onions, and leeks, and the flavonoids that fruits and vegetables are known for. Other phytochemicals like isofla- vones which are found in soy products help with the action of hormones in the body. The phytochemicals are called In- doles, and is present in cabbage and stimulates certain en- zymes which can change the effectiveness of oestrogen when it comes to breast cancer risks. Terpenes found in cherries and citrus fruits and the protease inhibitors in beans and soy- beans are both also responsible for enzyme stimulation. Some phytochemicals can interfere with the replication of DNA. Hot peppers contain capsaicin, and this chemical pre- vents carcinogens from harming the cellular DNA. Beans contain saponins, and these substances keeps cancer cells from multiplying by not allowing DNA replication. Other chemicals in plants can be a natural antibacterial, including allicin and is found in garlic. Proanthocyanidins are phytonu- trients found in cranberries, and they bind to the cells so that bacteria can not stick to the urinary tract as easily. 76

Getting the right phytonutrients, and in the right amounts, means eating a wide variety of foods including plenty of fresh fruits, beans, vegetables, and whole grains. The brighter and more vivid the color of a fruit or vegetable, the more phytochemicals that fruit or vegetable will contain. The rec- ommendations are that one should eat between five and nine servings of fruits and vegetables every single day, and should also include whole grains and beans in the diet frequently during the week. Eating a wide variety of foods, and exclud- ing sugar, highly processed foods, and alcohol from your di- et, will expose the widest variety of phytochemicals possible to protect the body. Phytochemicals are synthesized in plants that have been allowed to vine-ripen. Foods harvested green do not have these synthesized phytochemicals. Phytochemi- cals are synthesized in plants to protect the ripe plants from the damage caused by the ultraviolet rays from sunlight. These same chemicals have been proven to have a profound effect on the human immune system. Although in the past, phytonutrients in fruits and vegetables were classified as vit- amins, nowadays they are grouped according to their similar protective functions and similar chemicals characteristics. These groups are: 1. Terpenes, which are found in green foods, soy prod- ucts and grains, comprise one of the largest classes of phyto- nutrients. 2. Catenoids consist of bright yellow, orange and red plants pigments found in vegetables such as tomatoes, pars- ley, oranges, pink, grapefruit, spinach, and red pal oil. 3. Limonoids are a subclass found in citrus fruit peels. 4. Phytosterols occur in most plants, highly concentrated in the seeds of the plants. 5. Phenols are the blue, blue red and violet colorations seen in berries, grapes and purple eggplant. 77

6. Flavonoids are a phenol subclass and they enhance the effects of ascorbate-vitamin C. 7. Anthocyanidins or ―flavonals provide cross links that connect and strengthen the intertwined strands of colla- gen protein. 8. Catechins, Gallic Acids, differ slightly in chemical structure of other flavonoids, but share their chemoprotective properties. Catechins are found in green tea, and are thought to be responsible for the protective benefits of this beverage. 9. Isoflavones of this phenol subclass come from beans and other legumes and are a distant cousin of flavonoids. 10. Thiols are a sulphur-containing class of phytonutri- ents and are present in garlic and cruciferous vegetables. 11. Glycosylates are also found in cruciferous vegetables, and are powerful activators of liver detoxification enzymes. 12. Allylic Sulphides are a thiol subclass and is present in garlic, onions, leeks, shallots and chives. 13. Indoles are a subclass that interact with vitamin C, and are found in vegetables high in vitamin C. 14. Isoprenoids neutralize free radicals in a unique way. They grab free radicals and pass them off to other antioxi- dants. 15. Tocotrienols and Tocopherols naturally occur in grains and palm oils. 16. Lipoic Acid and Ubiquinone are important antioxi- dants that work to extend the effects of other antioxidants. 17. Foods and herbs that demonstrate the highest anti- cancer protection are: garlic, soybeans, cabbage, ginger, lico- ricey, and the umbelliferous vegetables like carrots, celery, cilantro, parsley, and parsnips. 18. Foods with a bit less protection include onions, flax, citrus, turmeric, cruciferous vegetables, solanaceous vegeta- bles such as tomatoes and peppers, brown rice and whole wheat. A measure of anti-cancer activity was also found in 78

oats, barley, mints, rosemary, thyme, oregano, sage, basil, cucumber, cantaloupe and berries. Obtaining maximum benefits of phytochemicals is as easy as including fruits, vegetables, spices and herbs in daily diet. For optimal health, eat at least 5 to 9 servings of fruit and vegetables and whole grains daily, and include soy foods regularly. Give those mighty nutrients, phytochemicals, the chance to do their job. Rheum Spiciforme, Leh (Ladakh); Science Citation: Surinder Kitchlu 79

Echinops Cornigerus, Leh (Ladakh); Science Citation: Surinder Kitchlu 80

Phytochemicals and Antioxidants: It seems like everywhere we look these days the word 'an- tioxidant' is being touted as absolutely the best thing to make us younger looking and more vital. But what are these things? Antioxidants are a class of phytochemicals and vita- mins; phytochemicals come from plants (phyto=plant). Anti- oxidants stop free radical oxidative processes (aging). Rust on metal is due to oxidation; the same thing happens inside our cells. When cells age enough, they die, in a process called apoptosis, which is triggered by certain changes of ag- ing. This is why the subject of 'antioxidants' has become so popular in the anti-aging movement. The major antioxidant produced by our own bodies is glutathione. We take other antioxidants (vitamins B, C, E, etc) to recy- cle the glutathione. In order to recycle the glutathione the vit- amin C and other antioxidants will actually accept the free radial itself-kind of like a relay runner handing off the button to the next runner. Maybe the fire brigade is a better illustra- tion, as the antioxidant's purpose is to quench free radicals, the fire is the free radical, the water is the antioxidant, and the firemen are the auxiliary antioxidants that replenish the water. Phytochemicals are made by plants as waste products (that's right, it's plant poop) and are classed into the follow- ing families: flavonoids, isoflavones (like phytoestrogens), isothiocyanates, monoterpenes, organosulfur compounds, saponins, and capsaicinoids. These are general antioxidants, cancer-preventers, anti-stiffening agents (for cell mem- branes), immune boosters, anti-cholesterol, glycaemic agents, anti-inflammatories, analgesics, and ANTI-APOPTOSIS. Good health has to do with getting a variety of all these phy- tochemicals, not so much in overdosing on certain ones (alt- 81

hough vitamin C is fairly safe to do so). The old saying that \"An apple a day keeps the doctor away.\" Well, the antioxi- dant properties of fruits and vegetables are what keep the body functioning properly. Plants help humans to poop but that wouldn't exactly be correct...it's usually the fiber of the plant that helps with that and it's the sugars that stimulate the peristaltic action of the intestines. At any rate, we need those phytochemicals for everything that happens in the body, be- cause they are vitamins, precursors, enzymes, hormones, etc. Phytochemicals are involved in so many processes which are absolutely mind-boggling. Beneficial effect of Phytochemicals: The newest thing in nutrition is phytochemicals - sub- stances that produce many of the beneficial effects associated with a diet that includes lots of fruits, vegetables, beans, and grains. If you‘ve been eating plant-based food all your life you‘ve been getting plenty of phytochemicals without know- ing it. The following are all phytochemicals: - Carotenoids, the pigments that make fruits and vege- tables orange, red, and yellow (dark green vegetables and fruits like kiwi contain these pigments, too, but green chlorophyll masks the carotenoids’ colors) - Thiocyanates, the smelly sulphur compounds that makes to turn up nose at the aroma of boiling cabbage - Daidzein and genistein, hormone-like compounds in many fruits and vegetables - Dietary fiber These and other phytochemicals, such as vitamins perform beneficial housekeeping in the body as they Keep bodies cells healthy 82

- Help prevent the formation of carcinogens (cancer- producing substances) - Reduce cholesterol levels - Help move food through intestinal tract The undeniable value of phytochemicals is one reason the U.S. Department of Agriculture/Health and Human Services Dietary Guidelines for Americans urges us to have at least five servings of fruits and vegetables and several servings of grains every day. Plants don‘t manufacture minerals; they ab- sorb them from the soil. Therefore, minerals aren‘t phyto- chemicals. List of plants containing phytochemicals: Vegetables Fennel Garlic Broccoli Tomato Fruits and nuts Acai Mangosteen Almond Maqui Berry Bilberry Noni Black Raspberry Olive Blackberry Orange 83

Blackcurrant Pomegranate Blueberry Red Raspberry Cranberry Sea Buckthorn Wild Strawberry Grape Wolfberry Guarana Hazelnut Medical plants Lungwort Comfrey Opium Poppy Passion Fruit Common Broom Echinacea Periwinkle Ginkgo Red Bryony Goat's Rue Valerian, Wintergreen Lesser Celandine Common herbs Rooibos Aloe Vera Rosemary American Ginseng 84

Clary Sage Sage Common Mallow Schizandra Cornsilk Stinging Nettle Dandelion Sweet Clover Ground Ivy Tea Hawthorn Wild Carrot Hop Wild Pansy Hyssop Woodruff Indian Cress Korean Ginseng Lemon Balm Milfoil Lemon Verbena Milk Thistle Marigold Red clover Beans and seeds Cacao Flaxseed Soy 85

Ephedra gerardiana, Leh; Science Citation: Surinder Kitchlu Arnebia guttata, Nyoma Ladakh, Science Citation: Surinder Kitchlu 86

Dietary phytochemicals shows a promise to chemopreven- tion. As cancer is a growing health problem around the world. Cervical cancer is commonest among women, fol- lowed by breast, esophagus, ovary, and stomach cancer. Among men most common are lung, bronchus, stomach, oral cavity, pharynx, larynx, prostrate, and rectum cancer. Syn- thetic anticancer drugs evoke severe side effects and in many cases patient may recover cancer but may die due side effects and severe immunosuppression. Several dietary phytochemicals are an alternative due to their various degree of antiproliferative and immunostimula- tory effects on various types of human cancers. This review will discuss the potentiality and promise of few plant derived secondary metabolites and there mode of actions in different human cancers. Facts and Figures WHO reported more than 10 million cases of cancer per year worldwide. In 2003, approximately 1,300,000 new cases were diagnosed, and more than 550,000 people died from cancer in US alone. As per National Cancer Registry Pro- gram of ICMR (1997), Chennai is the pocket of highest inci- dence of different cancers (stomach, cervix, ovary, prostrate, and lung) in India (Chaudhry and Luthra). According to Mudur (2005), some Indian cities shows world‘s highest can- cer incidence. For example, New Delhi for gall bladder can- cer in women, Wordha for mouth cancer, Pondicherry for tong cancer in men, and Kohima for nasopharyngeal cancer. Why Phtochemicals? Cancer is a multistage carcinogenic process where there is a net accumulation of a typical cells arising from excess pro- liferation, an insufficient apoptosis or a combination of the 87

two (Hetts, 1998). The conventional radiotherapy and chemotherapy with synthetic drugs evoke severe side effects including severe immunosuppression and in majority of cases patient may recover cancer but ultimately die due to infec- tious diseases and organ failure. Thus from therapeutic point of view, the best strategy is ― induced apoptosis in the neoplastic cell line without af- fecting the normal cells of the body (Fan et al., 1998). In this context, dietary phytochemicals are a potential alternative source of safer chemicals which are not only anticancerous but are also antioxidants, antidiabetic, antimutagenic, and other physiological benefits. Some of these common dietary bioactive secondary metobolites namely Curcumin, Resvera- trol, Sulforaphane, Styrylpyrone derivatives, Theasinensin-A, Theaflavin, Acacetin etc. show various degree of their indi- vidual antiproliferative effect and induction of apoptosis in various type of human cancers in various cytotoxic pathways. This review will deal with these phytochemicals and their mode of actions. Killing of Cancer cells Phytochemicals generally acts on apoptotic pathways. Apoptosis is a genetically controlled cellular suicide method by which unwanted cells are removed from the system with- out necrosis. Generally there are two pathways: Extrinsic pathway (Caspase-8 and 12) and Intrinsic pathway (Caspase- 9). The intrinsic pathway can be upregulated by treatment with drug like Cisplatin (Fulda et al., 1997), Doxorubicin and Methothrexate (Friesen et al., 1997) thus amplifying the caspase signal to bring apoptosis. The intinsic pathway is in- duced by ionizing radiation and also by chemotherapeutic drugs (Renatus et al., 2001) 88

In Intrinsic pathway, Cytochrome-C is released from the mitochondria to the cytosol by the action of external stimuli (Kluck et al., 1997). Released Cyt-C acts as a co-factor and interacts with Apaf-1 and procaspase-9 to form apoptosome. Apoptosome activates caspase-9 (Zou et al., 1997). Active casp-9 activates down stream (executioner) caspases like caspases like caspase-3,7 and 6 by limited proteolysis to fa- cilitate cell death (Earnshaw et al., 1999). Most of these phy- tochemicals work on this pathway. Phytochemicals and their mode of actions Curcumin (diferuloylmethane), the yellow pigment in rhizome of turmeric (Curcuma longa) brings apoptosis in several types of cancers including prostrate, breast, colon and leukemia through mitochondrial pathway caspase-8, BID cleavase, Cyt-C release and Caspase-3 activation. BCL-2 and BCL-XL are critical regulator of curcumin (Anto et al.,2002). Thesinensin-A from oolong tea induce apoptosis elevat- ing ROS production, release of Cyt-C and by induction of Caspase-9. Theaflavin (TF-1 & TF-2) effective on acute T- cell leukemia cause rapid induction of Caspase-3 but not caspase-1 (Pan et al.,2000) Sulforaphane (SFN) from criciferous plants like Brocco- li, Cabbage, Cauliflower, and their sprouts causes G2/M- phase arrest, increase apoptotic cell fraction in leukemia cell, markedly increase in P53 and Bax-expression (Fimognari et al.,2002) . SNF brings growth inhibition by inhibition of Cdk-4 and cyclin D1 resulting G1/S cycle arrest with a down expression of BCL-2 (Wang et al.,2004). 89

Oxytropis lapponica, Leh; Science Citation: Surinder Kitchlu Santolina chamaecyparissus L. – Cotton Lavender, Kashmir, Photo Courtsey: Surinder Kitchlu 90

Styrylprone derivatives (SPD) from Goniothalamus sp inhibits proliferation and induce apoptosis in MCF-7 cell line (breast cancer) by increasing BAX level and Caspase-9 which in turn activates executioner Caspase-7 to bring apop- tosis. (Alvin et al.,2003). Resveratrol, a phytoalexin red grape skin (Vitis vinifera) acts as an estrogen receptor (ER) agonist and suppress prolif- eration by ER independent pathway by inducing upregulation of P21 and brings growth arrest (Levenson et al.,2003). At high concentration inhibits Cyclin-D and CDK4 and upregu- lates P53 and P21 to bring apoptosis and in MCF-7 line by activating Caspase-9 increasing BAX, BAK and decreasing BCL-2 and BCL-XL (Kim et al.,2004). In recent years, natural bioactive products have attracted considerable attention as a new source of medicinal and ag- rochemical compounds. In several countries of southeast Asia, crude extracts from leaves and flowers of different Aglaia (family Meliaceae) plants are used in traditional med- icine (e.g., in Vietnam) for the treatment of inflammatory skin diseases and allergic inflammatory disorders such as asthma. The active compounds isolated from these plants are derivatives of rocaglamide. Rocaglamide and its naturally occurring congeners are tetrahydrobenzofurans that occur exclusively in members of the genus Aglaia. In the past, these natural products have attracted attention due to their strong insecticidal activity. More recently, cer- tain rocaglamide derivatives have also been found to have an inhibitory effect on the activity of the proinflammatory tran- scription factor NF-κB. Inflammatory diseases arise from in- appropriate activation of the immune system, leading to ab- normal expression of genes encoding inflammatory cytokines 91

and tissue-destructive enzymes. Many inflammatory genes are regulated at the transcriptional level by proinflammatory transcription factors, such as NF-κB and AP-1. During the immune response, Th cells produce various cy- tokines required for an efficient suppression of infections. Th1 cytokines IFN-γ and TNF-α promote cell-mediated im- munity, and Th2 cytokines IL-4, IL-5, IL-6, IL-10, and IL-13 promote humoral (Ab) immunity. However, uncontrolled ex- pression of these cytokines is dangerous and causes inflam- matory diseases. Dysregulation of IFN-γ and TNF-α produc- tion may contribute to the pathogenesis of many chronic in- flammatory diseases, including rheumatoid arthritis, diabetes, and hepatitis. Overexpression of IL-4 leads to atopic disor- ders, including allergen induced asthma, rhinoconjunctivities, and anaphylaxis. The expressions of IL-4, IFN-γ, and TNF-α are regulated by a number of inducible transcription factors, including NF-κB, AP-1 (Fos/Jun), and NF-AT. In resting T cells, NF-κB is sequestered into an inactive state by the cyto- plasmic inhibitor of NF-κB (IκB). T cell activation through TCR leads to the rapid activation of the IκB kinases (IKKs) via protein kinase C and results in phosphorylation, ubiquitylation, and subsequent degradation of IκB proteins, which allows nuclear translocation of NF- κB. In contrast, NF-AT family proteins are calcium- and cal- cineurin-regulated transcription factors. In resting T cells, NF-AT proteins are phosphorylated and reside in the cyto- plasm. T cell activation leads to activation of the Ca2+- dependent phosphatase calcineurin, resulting in rapid dephosphorylation of NF-AT and its translocation to the nu- cleus. T cell activation also induces the MAPKs, including ERKs, JNKs, and p38 that promote the synthesis, phosphory- lation, and activation of AP-1 transcription factors. MAPKs 92

have also been implicated in phosphorylation and thereby prevention of the nuclear localization of NF-AT. In addition, the NF-AT proteins are frequently found to act synergistically with AP-1 on composite promot- er/enhancer elements that contain adjacent NF-AT and AP-1 binding sites. Rocaglamides are potent immunosuppressive phytochemicals that suppress IFN-γ, TNF-α, IL-2, and IL-4 production in peripheral blood T cells at nanomolar concen- trations. Rocaglamides, at the doses that inhibit cytokine production, selectively inhibit the activity of NF-AT without impairing NF-κB and AP-1 activities. Derivatives of rocaglamide may serve as a new source of NF-AT-specific deliberately-induced clinical immunosuppression is used dur- ing bone marrow transplantation, organ transplantation, and in the treatment of auto-immune disease. Inhibition of intracellular calcineurin, a phosphatase that stimulates interleukin-2 expression during the normal T- lymphocyte immune response, has been a favorite target of immunosuppressive approaches. Cyclosporine and FK506 are the most notable of these calcineurin inhibitors, and have been used for many years as highly effective immunosup- pressants. More recent approaches use these agents in com- bination with others that do not target calcineurin. Thus, cur- rent immunosuppression regimens often include a combina- tion of agents including tacrolimus (Prograf, FK506), myco- phenolate mofetil (CellCept, MMF), sirolimus (Rapamune, Rapamycin), cyclosporine (Gengraf, Neoral, Sandimmune), azathioprine (Imuran) and others. Despite their effectiveness, clinical immunosuppressants have historically produced numerous side-effects. These in- clude risk of infection, nephrotoxicity, nausea, stomach up- 93

set, vomiting, diarrhea, high blood pressure, neurotoxicity, headaches, tremors, diabetogenicity, altered salt levels, numbness, cholesterol elevation, triglyceride elevation, ane- mia, reduced platelet count, rash or mouth sores, delayed wound healing, joint pain and others. In addition, patients on these immunosuppression regimens have a significantly higher risk of cancer than the general population, particularly lymphomas and skin cancer. Thus, there is significant inter- est in developing immunosuppressive approaches with less toxicity while maintaining sufficient immunosuppression and acceptable acute rejection rates. We became interested in immunosuppression research due to the studies on the gene RCAN1/ADAPT78, whose protein product (RCAN1) has been found to inhibit calcineurin. Cal- cineurin is a major mediator of calcium signaling and, as mentioned above, a target for effective immunosuppressants such as cyclosporine and FK506. In evaluating immunosup- pression, we considered a novel alternative approach based on the knowledge that phytochemicals can modulate gene expression. Specifically, we hypothesize that select phyto- chemicals can potentially be used to purposely modulate gene expression to gain clinical benefit. Even though it is now known that phytochemicals can modulate gene expres- sion, their use in treating genetically-defined clinically- related conditions has not been a focus. In order to use this approach, the genes that need to be modulated have to be clearly defined. In the case of clinical immunosuppression, it is IL-2 (and accompanying T-cell proliferation). That is, the activation of T-lymphocytes and their induction of IL-2 pro- duction are what sustain their proliferation during the im- mune response, and therefore underlie transplant allograft re- jection and autoimmunity. 94

Since our approach focuses on select phytochemicals, it has the major advantages of using phytochemicals with known health benefits. This is in contrast to existing toxic immunosuppressants, or future experimental ones with un- known long-term effects and usually high cost. By ―healthy, we mean that reports of a dietary agent's benefits to health have been published in respected peer-reviewed journals, and that the agent has been used for health benefit for a long pe- riod of time. Many studies have demonstrated the beneficial effects of phytochemicals from fresh fruits and vegetables including their prevention or delay of chronic degenerative diseases. These plants contain numerous and diverse nutrients in- cluding polyphenols, antioxidants, vitamins, phase 11 induc- ers (such as sulforaphane), and potent chemoprotectants that are thought to be major contributors to their healthy effects, and their individual nutrients are also known to modulate gene expression. Health benefits have been reported for all of the plant-based compounds that we have tested for their ef- fects on immunosuppression. To date, only a modest number of studies have investigat- ed the effects of select phytochemicals on T-cell suppression. Most notably, those that have have focused on curcumin, an anti-inflammatory and antioxidant component of turmeric. Interestingly, two curcumin studies report a strong suppres- sion of Tlymphocyte proliferation with near-identical inhibi- tory IC50 values of 3 uM . Based on these and other studies, we hypothesized that select phytochemicals can suppress T- lymphocyte proliferation both in vitro and in vivo. Our objec- tives were to test selected phytochemicals, including those previously untested, for their in vitro suppression of human T-cells and mouse splenocytes; to include selected phyto- 95

chemicals for in vivo analyses; and to assess the effect of cell culture on the recall of in vivo phytochemical effects. Addi- tionally, we included curcumin as a positive control to com- pare our results with studies from other laboratories. Com- bined, we believe that our approach of using select phyto- chemicals as clinical immunosuppressants is simple, healthy, and cost-effective inhibitors for the treatment of certain in- flammatory diseases. We have considered a novel ―rational gene targeting ap- proach for treating pathologies whose genetic bases are de- fined using select phytochemicals. We reason that one such potential application of this approach would be conditions requiring immunosuppression such as autoimmune disease and transplantation, where the genetic target is clearly de- fined; i.e., interleukin-2 and associated T-cell activation. Therefore, we hypothesized that select phytochemicals can suppress T-lymphocyte proliferation both in vitro and in vivo. The immunosuppressive effects of berry extract, curcumin, quercetin, sulforaphane, epigallocatechin gallate (EGCG), resveratrol, α-tocopherol, vitamin C and sucrose were tested on anti-CD3 plus anti-CD28-activated primary human T- lymphocytes in culture. Curcumin, sulforaphane, quercetin, berry extract and EGCG all significantly inhibited T-cell proliferation, and this effect was not due to toxicity. IL-2 production was also re- duced by these agents, implicating this important T-cell cy- tokine in proliferation suppression. Except for berry extract, these same agents also inhibited mouse splenic T-cell prolif- eration and IL-2 production. Subsequent in vivo studies re- vealed that quercetin (but not sulforaphane) modestly sup- pressed mouse splenocyte proliferation following supplemen- tation of BALB/c mice diets. 96

This effect was especially prominent if corrected for the loss of supplement ―recall as observed in cultured T-cells. These results suggest the potential use of these select phyto- chemicals for treating autoimmune and transplant patients, and support our strategy of using select phytochemicals to treat genetically-defined pathologies; an approach that we be- lieve is simple, healthy, and cost-effective. The heartwood of Dalbergia odorifera T. Chen.(Leguminosae) is a traditional Chinese medicine, known as jiangxiang in China. It has been used to treat blood disorder, ischemia, swelling, necrosis and rheumatic pain (1), indicating possible antiplatelet aggregation and anti- inflammatory activities. This plant had been reported to ef- fect inhibition of leukotriene biosynthesis in mammals (2), inhibition of prostaglandin biosynthesis (3), and anti- inflammatory activities (4). In our continuing search for an- tiallergic and anti-inflammatory agents from natural sources, we found that the methanolic extract of the heartwood of D.odorifera inhibited the release of β-glucuronidase and ly- sozyme from rat neutrophils, and the release of β- glucuroni- dase from rat mast cells. We did not determine the effect of the methanolic extract on the release of histamine from rat mast cells because fractions A-D affected the histamine assay system. From the methanolic extract of D.odorifera, we isolated three new compounds, (3R) - 4‘-methoxy-2‘,3, 7-trihydrox- yisoflavanone (11), 7-methoxy-3,3‘,4‘,6-tetrahydroxyflavone (18), and 2‘,7 -dihydroxy -4‘,5‘-dimethoxyisoflavone (22), together with twenty-two known compounds. Three new fla- vonoids, (3R)-4‘-methoxy-2‘, 3,7-trihydroxyisoflavanone (11), 7-methoxy-3,3‘,4‘,6-tetrahydroxyflavone (18), and 97

2‘,7-dihydroxy-4‘,5‘-dimethoxyisoflavone (22), were isolat- ed from the heartwood of Dalbergia odorifera T. Chen.(Leguminosae),together with twenty-two known com- pounds, (S)-4-methoxydalbergione (1), cearoin (2), medicar- pin (3), formononetin (4), sativanone (5), 3-hydroxy-9- methoxycoumestan (6), meliotocarpan A (7), isoliquirtigenin (8), stevein (9), liquiritigenin (10), 3‘,4‘,7- trihydroxyflavanone (12), butein (13),3‘-hydroxymelanettin (14), koparin (15), bowdichione (16), fisetin (17), melanettin (19), sulfuretin(20), 3‘-hydroxydaidzein (21), 3‘-o- methylviolanone (23), xenognosin B (24), and dalbergin (25). These flavonoids were evaluated in antiallergic and anti- inflammatory tests. The results showed that(S)-4- methoxydalbergione(1) and cearoin (2) exhibited antiallergic activity while(S)-4-methoxydalbergione(1),cearoin (2),butein (13), Koparin (15), bowdichione (16), 3‘-O-methylviolanone (23), and xenognosin B (24) showed significant anti- inflammatory activity. The present study examines the ef- fects of the extracts [petroleum etherCHCl3/MeOH(9:1) and MeOH], partially purified compounds from Guettarda acre- ana on the electrically induced contractions (E.C.I) of the iso- lated guinea pig ileum. The results of the experiments indi- cate that CHCl3/MeOH (9:1), MeOH extract, and the MeOH soluble part from CHCl3/MeOH extract tested at concentra- tions of 1.2, 2.5 and 5 µg/ml, dose dependently reduced the guinea pig ileum contractions. Furthermore, some partially purified fractions I-IV from the MeOH extract, each tested at the same concentrations of the extracts, and some pure com- pounds (6 * 10^-6, 3*10^-6, 1*10^-6 M ) isolated from the above fractions significantly reduced, in a dose dependent manner, the electrical contractions of the ileum. 98

The active compounds were identified as the known in- dole alkaloids strictosidic acid, lyalosidic acid, 5α- carboxystrictosidine, strictosidine, and sickingine, as well as the known quinic acid derivatives 5 - caffeoylquinic acid, 4,5-dicaffeoylquinic acid, and shikimic acid by spectral data. Two unknown quinovic acid glycosides and a new triterpenic glycosides and a new triterpenic glycoside, quinovic acid 3β- O- α-rhamnopyranosyl-(1→3)-( β-glucopyranosyl-(1→6)- β- glucopyranoside, were also isolated and their structures were established by NMR and MS data. From the rhizomes of Begonia heracleifolia six known cu- curbitacins (1-6) were isolated. Based on spectral data (1D and 2D 1H-, 13C-NMR, ESI- and CI-MS) the structures were established as cucurbiracin B (1), cucurbitacin D (2), 23,24- dihydro-cucurbitacin D (3), 23,24- dihydro-cucurbitacin F (4), 2-O-β-glucopyranosyl-cucurbitacin B (5), and 2-O-β- glucopyranosyl-cucurbitacin D (6). Four of them (3-6) were so far not reported as contituents of Begonia spp. Varyingly strong antiproliferative activity towards tumor and immune cells was observed for three compounds (1-3), due to differ- ent structural features. 99

Hypericum perforatum, Kashmir, Science citation: Surinder Kitchlu In continuation of an ethnobotanical study on plants used in traditional medicine in Mexico, we have selected Begonia heracleifolia Schltdl. & Cham. (Begoniaceae) for further phytochemical investigation. B. heracleifolia is a native spe- cies of humid Southern Mexico (1). Fresh rhizomes and peti- oles are used by the Zapotec Indians of Oxaca to treat wounds, local infections (such as acne, comedo, pustules, in- sect bites, stings), rheumatism, pain, and ―tumors (2). In a multiple screening, several fractions of the crude extract showed antibacterial and cytotoxic activity with noteworthy specific inhibition of cytokine-dependent cell lines. (3). Bio- activity-guided fractionation yielded six cucurbitacins (1-6); 100