Title of Invention

A PROCESS FOR ENHANCED IN VITRO PRODUCTION OF HYPERICIN FROM HYPERICUM HOOKERIANUM

Abstract

A process for the enhanced production of hypericin from call! and shoot cultures by the use of inducible synthesis of hypericin in cells and tissues, in cultures of Hypericum hookerlanum with exogenous supply of the hormone NAA, wherein, the calli and shoot cultures are obtained by inducing calli and growth from small pieces of H.hookerlanum shoot cultures, culturing in A callus and shoot cultures in callus and shoot induction medium respectively, for two weeks followed by subculture of the resultant callus and shoot for 6 weeks in callus and shoot proliferation medium respectively, followed by repeated sub-culturing of the calli and shoots in the same formulations at 6 weeks intervals for the continuous production of calli and shoots with red pigmentation for hypericin extraction.

Full Text

A process for the enhanced production of Hypericin, a secondary metabolite, from shoot and "callus cultures of Hypericum hookerianum using a novel culture in vitro. This invention relates to production of hypericin, a plant-specific, anti-retroviral and anti-HIV medicinal compound through a novel biotechnological process. In particular, this invention relates to production of hypericin from shoot and callus cultures of Hypericum hool There has been remarkable revival of interest in plant-derived drugs, cosmetics, flavours and such other natural products in very recent years. It is largely due to growing realization that synthetic alternatives have various side effects on human body. In fact, a green wave has swept across the entire world as a result of which any phyto-derivative is immediately accepted and appreciated. However, growth of the phyto-pharmaceuticals industry is largely hampered by the nonavailability of the quality planting materials, seasonal fluctuations, labour intensive, time consuming and often uneconomical traditional cultivation, extraction and utilization of the plant resumes. The explosive growth of human population in certain parts of the world, vertical increase in material needs and consequent injudicious/ unsustainable use of natural resources have together resulted in unprecedented depletion of forest resources and consequently extirpation and extinction of many a plant species in the tropics. As civilization encroaches upon the once virgin lands, the habitats of species get fragmented or degraded and extinction of species eventually results. Even cursory estimates suggest that at the current level of tropical deforestation, nearly 60,000 higher plants will disappear from the face of the earth by the middle of this century. Over exploitation of certain species beyond their regeneration in the wild has also resulted in the scarce or non-availability of them even for traditional medicinal preparations. Mitsui Petrochemical Co., Japan first demonstrated biotechnological intervention for value-added product making and diversification in 1984. A cell culture system developed from the plant Lithospermum erythrorhizon was commercialised for large-scale industrial production of shikonin, a natural product traditionally used in Japan for treatment of burns and hemorrhoid as well as for bright red eyes. After this remarkable feat, plant tissue and cell cultures have been the subject of intensive investigation the world over and production of such other plant-derived products as rosmarinic acid, ubiquinones, gingenosides and vanillin using tissue and cell cultures is either commercialised or in the pipeline for commercial production. Cells and tissues of many plants under controlled conditions of culture inside shake flask or bioreactors produce plant specific molecules but in trace quantities. However, very few cultures have been selected, manipulated or upgraded for scaled up production of the secondary metabolites to make the systems commercially viable. Such enhanced culture systems/ processes alone are patentable as they are known to synthesize / accumulate high concentrations of high value molecules as shikonin, berberin, rosmarinic acid etc. Not more than ten such systems/processes have been patented or tested for commercial worthiness/ feasibility. The present invention relates to the extraction of Hypericin (in vitro), which is chemically an aromatic polycyclic napthodianthrone with photodynamic activity and established antidepressant and antiretroviral activities including anti-HIV besides broad-spectrum antimicrobial activity. It is a plant-specific, high-value, low-volume medicinal compound of the species, Hypericum perforatum distributed in parts of Europe and America. There are many products based on hypericin that are currently available in the global market particularly in countries like US, UK and Germany. The market survey conducted in 1999 estimate the trade on hypericin products to US $ 570 l^lllions of which US market alone accounts for US $ 210 million and Germany $55 million. However, there is considerable variation in hypericin content in these products not only between manufacturers but also batch-to-batch variation in the same product. This could be attributed to a considerable extent, to the existing variation in hypericin content seen in natural and cultivated populations of H.perforatum across the globe as production of hypericin from the plants is highly regulated by several factors like seasonal changes, phase of plant growth, type of source material used for extraction and time of harvest. Since the value of 1.0 mg of hypericin in the retail market (Source: Sigma Chemical Co, USA) has increased from US $ 100.6 in 2003 to US $ 200.3 in 2004, obviously due to increased demand of the substance in medicinal and chemical applications, there is a need for an alternative production process to ensure scaling-up, consistency and uniformity in quality of the compound. The biotechnology based production system now developed through the use of shoot and callus cultures of Hypericum hool Tropical Botanic Garden and Research Institute, a pioneer in plant research in this country has now developed a novel process for the production of hypericin using a biotechnological process Involving shoot and callus cultures of Hypericum liookerianum and extraction of the compound from the shoot and callus tissues. The production of the compound was increased manifold In the shoot and callus cultures by feeding an auxin (Naphthalene acetic acid) to the culture medium. The content of active principle so produced is many times higher than that present in the natural plants. The present investigation reveals that without destructive harvesting of the plant species from the natural forests, sustainable production of the compound at the bench-level is possible to provide an alternative source to guarantee the survival of the species in nature and also to attract industrial attention for commercial production. But for the present investigation, the Indian plant, Hypericum fiool Furthermore, the novelty of the invention is that the process developed through judicious combination of salts and nutrients in the synthetic medium along with a critical concentration of the hormone NAA triggered manifold increase in hypericin production both the callus and shoot cultures of Hypericum lioof production of hypericin with potential commercial application through scale-up. This makes the process of producing enhanced quantities of hypericin interesting, novel and commercially significant. The following are the steps which are used in the the production of the natural compound hypericin in enhanced quantities: • Germination of seeds in basal medium: Surface sterlized seeds of H.hookerianum were inoculated into MS basal nutrient formulation having 1/2 strength concentrations of macrosalts (0.15% phytagel, pH 5.8) • Establishment, multiplication and maintenance of shoot cultures: Excised individual nodes of the 3-week old axenic seedlings were inoculated into MS nutrient formulation having full strength salts, B5 vitamins, 3% sucrose and kinetin, 0.5 mg/l (0.15% phytagel, pH 5.8) for multiple shoot bud initiation in 4 weeks and then the shoot buds transferred to the same formulation containing 1.0 mg/l kinetin for maximum shoot proliferation in 4 weeks. The multiplied shoots were sub-cultured in the same medium with 0.5 mg/l kinetin at four week intervals to raise ready stocks of shoots. As and when needed, the shoot clumps were sub-cultured in medium containing l.Omg/l kinetin and 0.20 mg/l naphthalene acetic acid to induce hypericin synthesis in the shoots. • Establishment of callus cultures: The shoot cultures of H.hookerianum were cut into small pieces (2-4mm) and cultured in MS formulation with full strength salts, B5 vitamins, 3% sucrose, napthalene acetic acid 2.0 mg/l, kinetin 0.2 mg/l and 0.15% phytagel, pH 5.8 to induce callus initiation. for two weeks followed by culture for 6 weeks in callus proliferation medium (I^S formulation with full strength salts, B5 vitamins, 3% sucrose, NAA 2.0 mg/l, 0.15% phytagel, pH 5.8) before extracting hypericin from the callus tissue. • Extraction of hypericin from callus and shoot cultures: a. 100 gm fresh calli are air dried, powdered and extracted in a soxhiet apparatus for 6-8 hrs with 50 ml ethyl alcohol. The extract is then filtered, dried and the residue is resuspended in 5 ml methanol for HPLC estimation. b. 100 gm fresh shoot cultures are initially extracted in a soxhiet apparatus for 3-4 hrs with 50 ml diethyl ether. The residue is dried and re-extracted in a soxhiet for 6-8 hrs with 50 ml ethyl alcohol. The extract is then filtered, dried and the residue is re-suspended in 5 ml methanol for HPLC estimation. The yield of hypericin from 100 gm (fresh wt) tissue each of shoot and callus cultures is 248 mg and 180 mg respectively. • Yield: 100 gm fresh wt of the callus on extraction gives 180 gm of the compound. The cost of 1.0 mg hypericin from Sigma Chemicals Co. USA being $ 200.3 in the year 2004, price of hypericin from 100 gm fresh wt of the callus as per the current retail market value is US $ 36054 (Rs. 16,22,430/-) and for shoots $ 49674 (Rs.22,35,330/-). However, the amount of hypericin from 100 gm dry wt of the callus is more (3.51 gm) than the shoots (3.10 gm). Starting from a single node of the axenic seedling, 100 gm of shoots could be raised under the culture conditions described in 20 weeks. Similarly starting from a single 4 mm segment of shoot culture, 100 gm of fresh calli could be raised in 24 weeks under the culture conditions described. The use of 0.2 mg/l NAA for shoot culture and 2.0 mg/l NAA for callus culture was critical to obtain hypericin-rich" tissues under 8 hr photoperiod in both the culture systems. Since large number of nodes and stem segments are available in seedling and shoot cultures, scale-up production has becomepossible. Statement of invention: A process for the enhanced production of hypericin from calli and shoot cultures by the use of inducible synthesis of hypericin in cells and tissues, in cultures of Hypericum hookerianum with exogenous supply of the hormone - NAA, wherein, the calli and shoot cultures are obtained by inducing calli growth from small pieces (2-4 mm)of H.hookerianum shoot cultures, culturing in a callus induction medium (MS formulation having full strength salts, B5 vitamins, 3% sucrose, naphthalene acetic acid (NAA) 2.0mg/l and kinetin 0.2 mg/I, 0.15% phytagel, pH 5.8) and shoot cultures in shoot induction medium (MS nutrient formulation having full strength salts, B5 vitamins, 3% sucrose and kinetin 0.5 mg/l, 0.15% phytagel, pH 5.8) for two weeks followed by subculture of the resultant callus for 6 weeks in callus proliferation medium (MS medium supplemented with 85 vitamins, '3% sucrose, NAA 2.0 mg/l, 0.15% phytagel, pH 5.8) and shoot cultures in shoot proliferating medium (MS medium supplemented with B5 vitamins, 3% sucrose and kinetin 1.0 mg/l, 0.15% phytagel, pH 5.8)with 0.2 mg/l NAA, followed by repeated sub-culturing of the calli and shoots in the same formulations at 6 weeks intervals for the continuous production of calli and shoots with red pigmentation for hypericin extraction. Extraction of hypericin from the callus cultures by air drying 100 gm fresh calli, powdering and extracting in a soxhiet for 6-8 hrs with 50 ml ethyl alcohol, filtering the extract, drying and re-suspending the residue in 5 ml methanol for HPLC estimation and in the case of shoot cultures, extraction by air drying 100 gm red pigmented fresh shoots, powdering, and extracting in a soxhiet apparatus for 3-4 hrs with 50 ml diethyl ether to remove chlorophyll pigments, drying the residue and re-extracting in a soxhiet apparatus for 6-8 hrs with 50 ml ethyl alcohol, filtering the extract, drying, re-suspending the residue in 5.0 ml methanol for HPLC estimation to obtain 248 mg and 180 mg hypericin respectively from shoot and callus cultures on gm fresh weight basis. Advantages: Hypericum perforatum, a traditional source of hypericin in Europe and USA is either destructively harvested in nature or cultivated for hypericin extraction. There is tremendous variation in the percentage distribution of hypericin obtained from the natural populations from different ecological conditions across the globe as the production of hypericin is highly regulated by such factors like seasonal changes, phase of plant growth, type of source material taken for extraction and the time of harvest. Therefore, production of hypericin through callus and shoot cultures using a novel media not only ensures uniformity and quality in hypericin content but also serves as a reliable and continuous source for the production of this natural compound. None of the Indian species of Hypericum are so far known to be a source of hypericin. The present investigations confirm that H.tiool The biotechnologlcal method of hypericin production helps reduce the labour intensive cultivation/natural harvest and the cost of production as solvent extraction from the flasks is more easily and readily achieved than from the various parts of the hardy field plants that carry a number of secondary tissues. The high concentration of hypericin in the shoots (3.10%) and callus (3.51%) cultures compared to the wild plant (2.28%) and easy subculture of shoots and calli to produce hypericin at will, will make the biotechnology-based production most attractive. The yield of the compound from 100 gm fresh weight of the shoots, calli in Hypericum hool The end product (hypericin) obtained in callus and shoot cultures is more or less pure and does not require additional purification steps employed for extraction from field-derived plants. As such the process is simple and very economical and the compound is natural and no synthetic step is involved. We claim: 1. A process for the enhanced production or nypericm rrom cam ana shoot cultures by the use of inducible synthesis of hypericin in cells and tissues, in cultures of Hypericum hookerianum with exogenous supply of the hormone NAA, wherein, the calli and shoot cultures are obtained by inducing calli growth from small pieces (2-4 mm)of H.hookerianum shoot cultures, culturing in a cal'us induction medium (MS formulation having full strength salts, B5 vitamins, 3% sucrose, naphthalene acetic acid (NAA) 2.0mg/l and kinetin 0.2 mg/l, 0.15% phytagel, pH 5.8) and shoot cultures in shoot induction medium (MS nutrient formulation having full strength salts, B5 vitamins, 3% sucrose and kinetin 0.5 mg/l, 0.15% phytagel, pH 5.8) for two weeks followed by subculture of the resultant callus for 6 weeks in callus proliferation medium (MS medium supplemented with B5 vitamins, 3% sucrose, NAA 2.0 mg/l, 0.15% phytagel, pH 5.8) and shoot cultures in shoot proliferating medium (MS medium supplemented with B5 vitamins, 3% sucrose and kinetin 1.0 mg/l, 0.15% phytagel, pH 5.8)with 0.2 mg/l NAA, followed by repeated sub-culturing of the calli and shoots in the same formulations at 6 weeks intervals for the continuous production of calli and shoots with red pigmentation for hypericin extraction. 2. A process as claimed in claim 1, wherein the extraction of hypericin from the callus cultures is by air drying 100 gm fresh calli, powdering and extracting in a soxhiet for 6-8 hrs with 50 ml ethyl alcohol, filtering the extract, drying and re-suspending the residue in 5 ml methanol for HPLC estimation and in the case of shoot cultures, extraction by air drying 100 gm red pigmented fresh shoots, powdering and extracting in a soxhiet apparatus for 3-4 hrs with 50 ml diethyl ether to remove chlorophyll pigments, drying the residue and re-extracting in a soxiilet apparatus for 6-8 lirs witln 50 ml etlnyl alcoliol, filtering the extract, drying, re-suspending the residue in 5.0 ml methanol for HPLC estimation to obtain 248 mg and 180 mg hypericin respectively from shoot and callus cultures on gm fresh weight basis. A process as claimed in claim 1, wherein the callus initiation medium consists of full strength MS nutrient formulation supplemented with B5 vitamins, 2.0 mg/l NAA, 0.2mg/l kinetin, 3% sucrose and 0.15% phytagel (pH 5.8) under 8 hr photoperiod (light:25-50 \i Em'^s'S 25±2 Degree Cent.). A process as claimed in claim 1, wherein the culture conditions for optimal induction of the said calli is obtained when initially 500 mg fresh wt of the shoot segments are cultured in 60 ml of growth medium (in 250 ml flasks) for two weeks under 8 hr photoperiod (light:25-50 ^ Em"^ s'\ 25±2 Degree cent.). A process as claimed in claim 1, wherein the production medium formulation for enhanced production of said compound from the said callus consists of full strength jvis nutrient formulation supplemented with B5 vitamins, 2.0 mg/l NAA, 3% sucrose and 0.15% phytagel (pH 5.8). A process as claimed in claim 1, wherein the culture conditions for enhanced production of the said compound from the said calli as claimed in claim 1, is obtained when the two week old calli in the growth medium is transferred to the production medium (60ml in 250 ml flask) and kept for 4 weeks under 8 hr photoperiod (light:25-50 ^ Em"^ s'\ 25± 2 Degree cent.). A process as claimed in claim 1, wherein the nutrient formulation for the initiation of shoot cultures consists of shoot initiation medium of MS nutrient formulation supplemented with B5 vitamins, 0.50 mg/l kinetin, 3% sucrose and 0.15% phytagel (pH 5.8) A process as claimed in claim 1, wherein the culture conditions for enhanced production of the said compound from the said shoots as ' claimed in claim 1, is obtained when 500 mg fresh wt of the shoots is cultured in 60 ml of growth medium (In 250 ml flasks) under 8 hr photoperiod (light:25-50 n Em"^ s"S 25± 2 Degree Cent.), A process as claimed in claim 1, wherein the production medium for enhanced production of the said compound from shoot cultures consists of full strength MS medium supplemented with 1.0 mg/l kinetin and 0.2 mg/l NAA. .A process as claimed in claim 1, wherein the culture conditions for enhanced production of the said compound from the said shoot cultures as claimed in claim 1, is obtained when 4 week old shoot cultures in the growth medium are transferred to the production medium 60 ml (in 250 ml flask) and incubated for 4 weeks under 8 hr photoperiod (light: 25-50 [i Em'^ s'S 25±2 degree cent). , A process as claimed in claim 1, wherein the process for the continuous production of the said compound as claimed in claim 1, is attained \Nhen after harvesting the shoot cultures, a portion of the shoot-nodal segments are excised and used to re-culture in the medium while the remaining shoot cultures are extracted with diethyl ether and ethanol using soxhiet apparatus following standard procedures. .A process as claimed in claim 1, wherein a process for the continuous production of the said compound is achieved when the callus tissues are sub-cultured at 6 week intervals without loss of production ability at least for 10 subculture cycles. ■ A process as claimed in claim 1, wherein the process for the continuous production of the said compound is by induction of synthesis and accumulation of hypericin in the shoot cultures through subculture at 4 week intervals in full strength MS nutrient formulation with B5 Vitamins, 1.0 mg/l kinetin, 0.2 mg/l naphthalene acetic acid (NAA) and 3% sucrose (0.15% phytagel, pH 5.8). , A simple process for the extraction of the compound from the calli in pure form as claimed in claim 1 without the obvious need for the removal of chlorophyll. , A process for the enhanced production hypericin from callus and shoot cultures as claimed in claim 1 and as described in the complete specification.

Documents:

1146-che-2004-abstract.pdf

1146-che-2004-claims.pdf

1146-che-2004-correspondnece-others.pdf

1146-che-2004-correspondnece-po.pdf

1146-che-2004-description(complete).pdf

1146-che-2004-form 1.pdf

1146-che-2004-form 19.pdf

1146-chenp-2004 abstract duplicate.pdf

1146-chenp-2004 claims duplicate.pdf

1146-chenp-2004 description (complete) duplicate.pdf