Plant natural products constitute the most diversified source of unique chemical structures, providing the basis for identification of new lead molecules that serve as starting point for novel drug discovery. India has a rich heritage of using plant-derived or so-called herbal medicines since ancient times. Due to unique agro-climatic   conditions
and physio-geographical features, the Himalaya region harbors high-value medicinal plants, which serve as the source of many life-saving drugs. Despite extensive use in Indian traditional medicine, information on active constituents and scientific evidence for the disease-curing mechanisms of most of the Himalayan medicinal plants are still elusive. This chapter provides a brief introduction to plant-based medicine—present scenario and future prospects, information on the traditional Indian system of medicine, plant biodiversity of the Himalaya, important medicinal plants of this region and their medicinal uses, conservation procedure, marketing strategies, and various approaches to sustainable production of Himalayan medicinal plants.

Hypericum species exhibit affluence of bioactive polyprenylated metabolites derived from various chemical nuclei including acyl- and benzoylphloroglucinol, flavonoid and xanthone ^[1]. Prenylated xanthones receive special attention due to their biological impact on human health. Their biosynthesis is achieved through two stages, (i) formation of the xanthone nucleus, (ii) decoration by side chains. Aromatic prenyltransferases catalyse the introduction of prenyl groups to an electron-rich aromatic substrate. Metabolic profiling of Hypericum sampsonii, whose extracts are used in TCM to treat swellings, burns, backache and in Taiwan as antitumor drug, showed abundance of pharmacologically active prenylated xanthones. Patulone inhibits the exogenous platelet activating factor, induces hypotension in mice, and inhibits COX-1 enzyme ^[2]. Hypericumxanthones ^[3] and Hyperixanthone A ^[4] are antibacterial metabolites from H. sampsonii.

This work presents the complete elucidation of the biosynthetic pathway of patulone. Two prenyltransferases from H. sampsonii, which are responsible for the decoration of 1,3,6,7-tetrahydroxyxanthone to produce patulone, were studied at the gene level. Constructs of pECS-URA harbouring the individual genes were created for yeast expression. Activity screening of the recombinant proteins against potential substrates from different chemical classes revealed their narrow substrate specificity, the strict regiospecificity to C-8 of the 1,3,6,7-tetrahydroxyxanthone skeleton and absolute dependence on Mg²⁺ ions. The sequential prenylation of 1,3,6,7-tetrahydroxyxanthone was proved through determination of the kinetic parameters. The product of HsPT8PX is used as substrate for HsPTpat to form the gem-diprenylated patulone. When fused to YFP, both enzymes showed subcellular localization to chloroplasts. Our results inaugurate a novel chemo-enzymatic production approach for pharmacologically important trace natural products.

Pyrus pyrifolia (Asian pear) cell cultures respond to yeast extract (YE) treatment by accumulating benzoate-derived biphenyl phytoalexins, namely, noraucuparin and aucuparin. Biphenyl phytoalexins are defense-marker metabolites of the sub-tribe Malinae of the family Rosaceae. The substrates for biphenyl biosynthesis are benzoyl-CoA and malonyl-CoA, which combine in the presence of biphenyl synthase (BIS) to produce 3,5-dihydroxybiphneyl. In the non-β-oxidative pathway, benzoyl-CoA is directly derived from benzoic acid in a reaction catalyzed by benzoate-CoA ligase (BZL). Although the core β-oxidative pathway of benzoic acid biosynthesis is well-understood, the complete cascade of enzymes and genes involved in the non-β-oxidative pathway at the molecular level is poorly understood. In this study, we report the detection of benzaldehyde dehydrogenase (BD) activity in YE-treated cell cultures of P. pyrifolia. BD catalyzes the conversion of benzaldehyde to benzoic acid. BD and BIS activities were coordinately induced by elicitor treatment, suggesting their involvement in biphenyl metabolism. Changes in phenylalanine ammonia-lyase (PAL) activity preceded the increases in BD and BIS activities. Benzaldehyde was the preferred substrate for BD (K_m = 52.0 μM), with NAD⁺ being the preferred co-factor (K_m = 64 μM). Our observations indicate the contribution of BD towards biphenyl phytoalexin biosynthesis in the Asian pear.

Extracts of the medicinal plant Hypericum perforatum are used to treat depression and skin irritation. A major API is hyperforin, characterized by sensitivity to light, oxygen and temperature. Total synthesis of hyperforin is challenging and its content in field-grown plants is variable. We have established in vitro cultures of auxin-induced roots, which are capable of producing hyperforin, as indicated by HPLC-DAD and ESI–MS analyses. The extraction yield and the productivity upon use of petroleum ether after solvent screening were ∼5 mg/g DW and ∼50 mg/L culture after six weeks of cultivation. The root cultures also contained secohyperforin and lupulones, which were not yet detected in intact plants. In contrast, they lacked another class of typical H. perforatum constituents, hypericins, as indicated by the analysis of methanolic extracts. Hyperforins and lupulones were stabilized and enriched as dicyclohexylammonium salts. Upon up-scaling of biomass production and downstream processing, H. perforatum root cultures may provide an alternative platform for the preparation of medicinal extracts and the isolation of APIs.

The active medicinal constituents in Hypericum perforatum, used to treat depression and skin irritation, include flavonoids and xanthones. The carbon skeletons of these compounds are formed by chalcone synthase (CHS) and benzophenone synthase (BPS), respectively. Polyclonal antisera were raised against the polyketide synthases from Hypericum androsaemum and their IgG fractions were isolated. Immunoblotting and immunotitration were used to test the IgGs for crossreactivity and monospecificity in H. perforatum leaf protein extract. Immunofluorescence localization revealed that both CHS and BPS are located in the mesophyll. The maximum fluorescence levels were observed in approx. 0.5 and 1 cm long leaves, respectively. The fluorescence intensity observed for CHS significantly exceeded that for BPS. Using histochemical staining, flavonoids were detected in the mesophyll, indicating that the sites of biosynthesis and accumulation coincide. Our results help understand the biosynthesis and underlying regulation of active H. perforatum constituents.

Pear (Pyrus communis) is an economically important fruit crop. Drops in yield and even losses of whole plantations are caused by diseases, most importantly fire blight which is triggered by the bacterial pathogen Erwinia amylovora. In response to the infection, biphenyls and dibenzofurans are formed as phytoalexins, biosynthesis of which is initiated by biphenyl synthase (BIS). Two PcBIS transcripts were cloned from fire blight-infected leaves and the encoded enzymes were characterized regarding substrate specificities and kinetic parameters. Expression of PcBIS1 and PcBIS2 was studied in three pear cultivars after inoculation with E. amylovora. Both PcBIS1 and PcBIS2 were expressed in ‘Harrow Sweet’, while only PcBIS2 transcripts were detected in ‘Alexander Lucas’ and ‘Conference’. Expression of the PcBIS genes was observed in both leaves and the transition zone of the stem; however, biphenyls and dibenzofurans were only detected in stems. The maximum phytoalexin level (~110 μg/g dry weight) was observed in the transition zone of ‘Harrow Sweet’, whereas the concentrations were ten times lower in ‘Conference’ and not even detectable in ‘Alexander Lucas’. In ‘Harrow Sweet’, the accumulation of the maximum phytoalexin level correlated with the halt of migration of the transition zone, whereby the residual part of the shoot survived. In contrast, the transition zones of ‘Alexander Lucas’ and ‘Conference’ advanced down to the rootstock, resulting in necrosis of the entire shoots.

Biphenyls and dibenzofurans are the phytoalexins of the Malinae involving apple and pear. Biosynthesis of the defence compounds includes two O-methylation reactions. cDNAs encoding the O-methyltransferase (OMT) enzymes were isolated from rowan (Sorbus aucuparia) cell cultures after treatment with an elicitor preparation from the scab-causing fungus, Venturia inaequalis. The preferred substrate for SaOMT1 was 3,5-dihydroxybiphenyl, supplied by the first pathway-specific enzyme, biphenyl synthase (BIS). 3,5-Dihydroxybiphenyl underwent a single methylation reaction in the presence of S-adenosyl-l-methionine (SAM). The second enzyme, SaOMT2, exhibited its highest affinity for noraucuparin, however the turnover rate was greater with 5-hydroxyferulic acid. Both substrates were only methylated at the meta-positioned hydroxyl group. The substrate specificities of the OMTs and the regiospecificities of their reactions were rationalized by homology modeling and substrate docking. Interaction of the substrates with SAM also took place at a position other than the sulfur group. Expression of SaOMT1, SaOMT2 and SaBIS3 was transiently induced in rowan cell cultures by the addition of the fungal elicitor. While the immediate SaOMT1 products were not detectable in elicitor-treated cell cultures, noraucuparin and noreriobofuran accumulated transiently, followed by increasing levels of the SaOMT2 products aucuparin and eriobofuran. SaOMT1, SaOMT2 and SaBIS3 were N- and C-terminally fused with the super cyan fluorescent protein and a modified yellow fluorescent protein, respectively. All the fluorescent reporter fusions were localized to the cytoplasm of Nicotiana benthamiana leaf epidermis cells. A revised biosynthetic pathway of biphenyls and dibenzofurans in the Malinae is presented.

Biosynthesis of phytoalexins is a plant defence strategy against pathogens. Shoots of the apple (Malus × domestica) cultivar ‘Holsteiner Cox’ formed biphenyls and dibenzofurans when inoculated with the fire blight bacterium, Erwinia amylovora. The phytoalexins were only present in the transition zone of stems, whereas the leaves were devoid of the defence compounds. The scaffold of the phytoalexins is formed by biphenyl synthase (BIS), a type III polyketide synthase. In apple, BIS is encoded by a gene family, members of which fall into four subfamilies. Representative BIS cDNAs were cloned from fire blight-infected shoots of ‘Holsteiner Cox’ and functionally expressed. The preferred starter substrates were benzoyl-CoA and salicoyl-CoA, leading to the formation of 3,5-dihydroxybiphenyl and 4-hydroxycoumarin, respectively, in the presence of malonyl-CoA as extender molecule. The four subfamilies were differentially regulated after inoculation of shoots with E. amylovora. The BIS3 gene was expressed in stems, with maximum transcript levels in the transition zone. The BIS3 protein was immunochemically localized to the parenchyma of the bark. Dot-shaped immunofluorescence was restricted to the junctions between neighbouring cortical parenchyma cells. Leaves contained transcripts for BIS2 which, however, were not translated into immunodetectable BIS protein. The understanding of phytoalexin metabolism may aid in improving apple resistance to fire blight.

Although a number of plant natural products are derived from benzoic acid, the biosynthesis of this structurally simple precursor is poorly understood. Hypericum calycinum cell cultures accumulate a benzoic acid-derived xanthone phytoalexin, hyperxanthone E, in response to elicitor treatment. Using a subtracted complementary DNA (cDNA) library and sequence information about conserved coenzyme A (CoA) ligase motifs, a cDNA encoding cinnamate:CoA ligase (CNL) was isolated. This enzyme channels metabolic flux from the general phenylpropanoid pathway into benzenoid metabolism. HcCNL preferred cinnamic acid as a substrate but failed to activate benzoic acid. Enzyme activity was strictly dependent on the presence of Mg²⁺ and K⁺ at optimum concentrations of 2.5 and 100 mm, respectively. Coordinated increases in the Phe ammonia-lyase and HcCNL transcript levels preceded the accumulation of hyperxanthone E in cell cultures of H. calycinum after the addition of the elicitor. HcCNL contained a carboxyl-terminal type 1 peroxisomal targeting signal made up by the tripeptide Ser-Arg-Leu, which directed an amino-terminal reporter fusion to the peroxisomes. Masking the targeting signal by carboxyl-terminal reporter fusion led to cytoplasmic localization. A phylogenetic tree consisted of two evolutionarily distinct clusters. One cluster was formed by CoA ligases related to benzenoid metabolism, including HcCNL. The other cluster comprised 4-coumarate:CoA ligases from spermatophytes, ferns, and mosses, indicating divergence of the two clades prior to the divergence of the higher plant lineages.

Fire blight, caused by the bacterium Erwinia amylovora, is a devastating disease of apple (Malus × domestica). The phytoalexins of apple are biphenyls and dibenzofurans, whose carbon skeleton is formed by biphenyl synthase (BIS), a type III polyketide synthase. In the recently published genome sequence of apple ‘Golden Delicious’, nine BIS genes and four BIS gene fragments were detected. The nine genes fall into four subfamilies, referred to as MdBIS1 to MdBIS4. In a phylogenetic tree, the BIS amino acid sequences from apple and Sorbus aucuparia formed an individual cluster within the clade of the functionally diverse type III polyketide synthases. cDNAs encoding MdBIS1 to MdBIS4 were cloned from fire-blight-infected shoots of apple ‘Holsteiner Cox,’ heterologously expressed in Escherichia coli, and functionally analyzed. Benzoyl-coenzyme A and salicoyl-coenzyme A were the preferred starter substrates. In response to inoculation with E. amylovora, the BIS3 gene was expressed in stems of cv Holsteiner Cox, with highest transcript levels in the transition zone between necrotic and healthy tissues. The transition zone was the accumulation site of biphenyl and dibenzofuran phytoalexins. Leaves contained transcripts for BIS2 but failed to form immunodetectable amounts of BIS protein. In cell cultures of apple ‘Cox Orange,’ expression of the BIS1 to BIS3 genes was observed after the addition of an autoclaved E. amylovora suspension. Using immunofluorescence localization under a confocal laser-scanning microscope, the BIS3 protein in the transition zone of stems was detected in the parenchyma of the bark. Dot-shaped immunofluorescence was confined to the junctions between neighboring cortical parenchyma cells.