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7.6 Chemical constituents 95
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Prakash and Bisht (2010) reported variation in respect of isoenzyme and
polypeptides in seeds of P. hexandrum having two and three leaves. The material
was collected from two populations one growing at Harkidun (3000 m), district
Uttarkashi, and the other in Valley of Flowers (3300m), district Chamoli, and cultivated at lower altitude Pothivasa (2200 m), district Rudraprayag of Uttarakhand.
Several low molecular polypeptides were prominent in all the populations. While
the presence of high molecular weight polypeptides was observed only in naturally
grown population having three leaves, some specific bands of isoenzyme were
observed in population cultivated at lower altitude.
Wani et al. (2012) assessed methanolic extracts of the two medicinal plants viz.
Rheum emodi and P. hexandrum for phytochemical components. The results
revealed that both the plant extracts contained glycosides, flavonoids, saponins,
and terpenes. Alkaloids were present at low quantity in R. emodi but were absent
in P. hexandrum. The absences of carbohydrates were detected in both the extracts.
Proteins were present in low quantity in R. emodi but abundantly present in
P. hexandrum.
The Indian P. hexandrum is superior to its American species, namely,
P. peltatum, in terms of higher podophyllotoxin content (4% in the dried roots in
comparison to only 0.25% for P. peltatum)(Hollithuis, 1988). Chaudhry et al.
(2014) investigated Podophyllum species and revealed presence of a number of com-
pounds like podophyllin, podophyllotoxin, quercetin, 4-demethylpodophyllotoxin,
podophyllotoxin glucoside, 4-dimethyl podophyllotoxin glucoside, kaempferol,
icropodophylotoxin, deoxypodophyllotoxin, picropodophylotoxin, sopicropodophyllone, 4-Methyl deoxypodophyllotoxin,-peltatin and S- peltatin. Nag et al.
(2013) reported that rhizomes and roots of P. hexandrum contain antitumor lignans
such as podophyllotoxin, 4-dimethyl podophyllotoxin, and podophyllotoxin 4-Oglucoside (Tyler et al., 1988; Broomhead and Dewick, 1990). Of these lignans,
podophyllotoxin is the most important for its use in the semisynthesis of anticancer
drugs, etoposide and teniposide (Issel et al., 1984). Podophyl lotoxin content of
Himalayan Mayapple is quite high (4.3%) compared to that of P. peltatum
(0.25%), the most common species in the America (Jackson and Dewick, 1984).
Hameed et al. (2014) had done comparative chemotaxonomic to investigate the
phylogenetic relationship of different acce ssions within the Podophyllum species.
Chemical profiles demonstrated that all P. hexandrum accessions collected from
different geographical regions are chemically diverse. Chemotaxonomic data
showed that chemical characters of the investigated species were able to generate
essentially the same relationship as revealed by RAPD analysis. The study has
revealed that maximum amount of the podophyllotoxin (5.97%) and podophyllotoxin b-D glycoside (5.72%) was present in the Podophyllum population collected
from Keller (Shopian) and Khilanmarg (Gulmarg) area of Jammu and Kashmir,
respectively. Mengfei et al. (2012) explained biochemical composition and antioxidant capacity of extracts from P. hexandrum rhizome. The rhizome extracts had
greater antioxidant capacity than the petiole extracts in DPPH and FRAP assays.
About 16 kinds of main reactive oxygen components were identified in the extracts.
96 CHAPTER 7 Podophyllum hexandrum
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Podophyllum herb contains 4.3% podophyllotoxin on a dry weight basis and it
contains three times more podophyllotoxin than in American species P. peltatum
(Fay and Ziegler, 1985; Nag et al., 2013). Podophyllotoxin content in
P. hexandrum has been reported between 1.03 and 6.13% in 30 different geographical sources from Himachal Pradesh and Ladakh (Singh et al., 2018).
7.7 Molecular advancements
There is urgent need to conserve genetic diversity of this valuable medicinal plant,
which may become extinct if reckless exploitation continues. Estimation of the level
and distribution of genetic variation in endangered species is a primary objective
implementation of conservation programs (Fritsch and Rieseberg, 1996). Therefore,
it is necessary to evaluate the genetic variation from different regions for
identification of elite germplasm with high genetic variability that can be used in
conservation. Chaudhary et al. (2014) observed that Podophyllum is becoming
rare and is at the risk of danger for being extinction. This exerted huge pressure
on the population may result in the extinction of species. So efforts should be
made to conserve germplasm of such valued species. For this certain protective measures should be taken. Exploitation in wild should be prohibited at Government level
in order to conserve this plant in its natural habitat. Xiao et al. (2006) said that
estimate of genetic diversity could provide a basis for conservation and utilization
of the endangered P. hexandrum. The results of the study from Western Sichuan
Province, China, showed that there is low genetic diversity at the species level
and that genetic differentiation among populations was more obvious than within
populations. Based on field survey of seven natural populations, they found that
the habitats of all populations have been destroyed by heavy deforestation and extensive habitat loss over the past few decades. These factors, together with overcollecting, have led to a decreasing in population size and subsequent inbreeding
depression. They also suggested initiating management program of ex situ conservation of the species. Their study also revealed that some popul ations harbor specific
locally adapted genotypes that are suited to particular environments. Artificially
propagated plants recruited from local seed sources are more likely to exhibit
increased fitness over nonlocal genotypes in particular environments. Consequently,
they suggest that care should be taken to separate seedlings from different populations and reintroduce seedlings only into their original parental localities. With combined and sustained efforts, they hope that the genetic diversity of the important
medicinal and endangered species of P. hexandrum in weste rn Sichuan Province
will be guaranteed.
P. hexandrum is described as self-incompatible but some researchers believe that
colonies in the wild may come from single seedling. Thus, one genotype grows in
clonal patches (Laverty and Plowright, 1988). In contrast, Policansky (1983)
reported that Mayapple colonies comprised more than one genotype and
7.7 Molecular advancements 97
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interpopulation crosses. This evidence suggests that Mayapple is at least partially
self-incompatible. Siddique et al. (1990) described the P. hexandrum
(2n ¼ 2x ¼ 12) for first time and found the haploid set comprise one metacentric,
three submetacentric, and two acrocentric chromosomes. On the basis of studies
on chromosome component of P. hexandrum from actively dividing root tip cells
of germinating seed she also revealed that the chromosomes could be grouped
into six duplets on the basis of overall length, centromeric position, and details of
NOR (nucleolar organizing region). Metaphase chromosome in P. hexandrum
(2n ¼ 12) is large in size, indicating toward a large genome size, which is found
to be 32.25 pg (2C content), using Vicia faba L. ‘Inovec (2c ¼ 26.90) as internal
standard (Dolezel et al., 2007). Nag and Rajkumar (2011) found the karyotype formula 6m þ 2m þ 2stþ 2t for P. hexandrum with secondary constriction in the
chromosomes 1 and 7. Callus tissues of P. hexandrum have unique potential of
generating variation in in vitro (Kumar and Mathur, 2004). Arumugam and Bhoj-
wani (1994) reported stability in chromosome number in 18-month-old callus cul-
ture of P. hexandrum with all cells showing the diploid chromosome number,
while both numerical and structural variation were observed by them in 3-yearold calli. The haploid set comprised of one metacentric, three submetacentric, and
two acrocentric chromosomes, with the metacentric chromosomes being the longest
and the acrocentric ones being the smallest in the complement; four chromos omes
has secondary constrictions. Numerical chromosome variation during callus culture
is evidently attained by some kind of endore-duplication and anaphase nondisjunction (Mukhopadhyay and Sharma, 1990). Genome multiplication is affected by
certain spindle anomalies giving rise to cells with abnormal chromosome numbers,
the frequency of which increases with time as a result of multiplication of division
errors. Mixoploid nature of the source explants and culture conditions have also been
suggested as some of the other causes leading to chromosomal variation in in vitro
(Phillips et al., 1994). RAPD markers have been used to differentiate P. hexandrum
populations from Chamba and Kullu district of Himachal Pradesh (Sharma et al.,
2000a,b) and in discriminating seven P. peltatum accessions collected from three
different sites in Lafayette County, Mississippi (Lata et al., 2002). Habitat fragmentation poses major threats to endangered plant species by reduc ing population size
and increasing geographic isolation (Young et al., 1996). The increased risk of
extinction of many endangered species including medicinal is often associated
with small and isolated populations, population genetics is highly relevant to development of conservation strategies of endangered species (Ellstrand and Elam, 1993;
Ottewell et al., 2015). Genetic variability has also been reported in P. hexandrum.
Singh et al. (2018) screened population of the P. hexandrum from different
geographical locations of Himachal Pradesh and Ladakh and observed considerable
variations in morphological characters viz., plant height, leaf shape, fruit weight, and
color. At least four distinct morphological variants with 1, 2, 3, and 4 leaves have
been reported (Purohitet et al.,1999). Polypeptide patterns and esterase enzyme analysis have indicated the existence of high inter- and intrapopulation variation in
98 CHAPTER 7 Podophyllum hexandrum
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P. hexandrum from the Gharwal Himalayas (Bhadula et al., 2000). Based on high
active ingredient content the HFRI has identified the superior chemo types of
P. hexandrum by screening different populations from Himachal Pradesh and
Ladakh. Purohit et al. (1999) also recorded significant variation in podophyllotoxin
content among different populations from Himachal Pradesh and Ladakh. Populations of P. hexandrum, collected from alpine region, have been observed highest
podophyllotoxin content. Substantial decrease in podophyllotoxin and resin contents
was observed when plants collected from higher altitudes were planted at low altitudes (Sharma et al., 2000a,b), whereas minor difference in podophyllotoxin content
of P. hexandrum was observed between wild plants and those cultivated in the region
of natural habitat (Prasad, 2000).
Further, the availability of genomics, transcriptomics, and metabolomics
resources can provide a powerful tool for identification, validation, characterization,
and functional analyses of molecular components in order to infer their role in regulation of metabolic processes in plant species. Kumar et al. (2015, 2016, 2017, 2018)
identified and validated crucial molecular components (pathway genes, transcription
factors, ABC transporters, and miRNAs) contributing to podophyllotoxin biosynthesis machinery in P. hexandrum. The research work laid the foundation for future
research aimed to escalate the production of bioactive compounds in Podophyllum
species and eventually will be beneficial for the industries and society.
7.8 Good agricultural and collection practices
Within the overall context of quality assurance, the WHO guidelines on good agricultural and collection practices (GACPs) for medicinal plants are primarily
intended to provide general technical guidance on obtaining medicinal plant materials of good quality for the sustainable production of herbal products classified as
medicines. They apply to the cultivation and collection of medicinal plants,
including certain postharvest operations. Raw medicinal plant materials should
meet all applicable national and/or regional quality standards.
The main objectives of these guidelines are as follows:
• To contribute to the quality assurance of medicinal plant materials used as the
source for herbal medicines, which aims to improve the quality, safety and
efficacy of finished herbal products.
• To guide the formulation of national and/or regional GACP guidelines and GACP
monographs for medicinal plants and related standard operating procedures.
• To encourage and support the sustainable cultivation and collection of medicinal
plants of good quality in ways that respect and support the conservation of
medicinal plants and the environment in general.
Extraction of the whole P. hexandrum plant in order to harvest the roots and
rhizome, containing the useful drug, Indian Podophyllum, has led to a decline of
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this species and even disappearance in some regions. It is variously considered as
endangered and critically endangered. It has declined considerably as a result of
exploitation to meet the increasi ng demands of the pharmaceutical industry. Export
of this species from I ndia has been prohibited, although illegal removal continues.
Medicinal plants are valued for their various active ingredient contents w hich are
used by the pharmaceutical companies for various formulations. Therefore, it is of
paramount importance that first of all we should identify the superior stock (high
active ingredient contents) from the natural condition and then multiply the same
for commercial cultivation. Development of cost-effective propagation methods
will further help to mass multiplication of elite planting material thus making
sure for the availability of quality planting stock to go for commercial cultivation
of the species. Because the spe cies is already endangered, and exploitation of its
underground parts continues to exceed the rate of natural regeneration, it needs
immediate attention for conservation. In an endeavor to d evelopment of the species
the Institute has made an effort to develop appropriate propagation methods and
identified the superior chemo types of P. hexandrum by screening different popu-
lations from Himachal Pradesh and Ladakh Valley (J&K). Various other institute
and agencies are also actively involved in development of agro-techniques of the
species.
7.8.1 Soil and fertilization
The enrichment of soil with nutrients is one of the most important tools to obtain
satisfactory productions (Carrubba and Alessandra, 2015). The practice of use of fertilizers is also growing in medicinal and aromatic plant species. The use of fertilizers
was defined often indispensable in order to obtain large yields. At the same time,
however, the use of correct types and quantities of fertilizers was termed necessary.
Generally an increased level of nutrients induces an enhancement of plant biomass,
but when the goal of cultivation is different from herbage yield, i.e., when a special
plant parts, i.e., seeds, roots, flowers, are of interest, or when the quality features are
especially important, the outcome of fertilization may be dramatically different. A
fine-tuned fertilization practice is therefore necessary, and forms, rates, and times
of distribution of fertilizers must be accurately planned and managed. Nutrient
availability in soils is related to several soil characters, both physical and chemical.
Soil reaction (pH) and parent materia l are the major factors moderating the content
and availability of mineral elements in the soil (Carrubba and Alessandra, 2015).
In natural condition, P. hexandrum has been found growing on open slopes and
under forest covers, along sides of Nallah, along the borders of cultivated fields and
species prefers moist habitat. The soil in natural habitat of the species is generally
black and rich in organic matters (Sharma and Sharma, 2018; Singh et al., 2018).
Under cultivated conditions, application of farmyard manures and humus increases
plant growth and yield. Mixing of well-rotted and sieved farmyard manure should be
done at the end of October or the beginning of November. Generally 50e60 quintal
farmyard manure is required for 1 hectare of land (Singh et al., 2018; Sharma and
Singh, 2014).
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7.8.2 Propagation methods
Nursery Raising and Management: Generally light and well-drained soils are
good for nursery raising. Before sowing of seeds, the beds should be properly
ploughed and be mixed with farmyard manures at 04 kg/m
sown during the month of SeptembereNovember and start germinating by Marche
April. Regular watering (at least twice/week) and weeding is required for proper and
healthy growth of nursery seedlings. Owing to its slow growth the seedlings become
ready for transplantation in the next year (Sharma and Singh, 2014).
Propagation Methods: P. hexandrum Royle can be propagated through seeds
and rhizomes (Nautiyal and Nautiyal, 2003; Qazi et al., 2011; Sharma and Singh,
2014; Sharma and Sharma, 2018). Seeds show erratic and poor germination under
natural conditions. Sometimes the seeds germinate after remaining dormant for 1
or 2 years. The main reason for poor seed germination seems to be postharvest
care of seeds. Nautiyal et al. (1987) observed no germination in seeds extracted
from fresh berries; however, germination was recorded when fresh berries were
used as such for germination. Seeds washed with water also showed better germination than unwashed seeds (Bhadula et al., 2000). Purohit and Nautiyal (1988) found
inhibitory effect of cotyledons on plumule development in P. hexandrum. Bhadula
et al. (2000) observed inhibitory effect of fruit pulp on seed germination and removal
of seed coat was found helpful in enhancing seed germination. The propagation of
species through seeds and rhizomes is described below.
2
. Seeds are generally
Mature seed capsule of P. hexandrum.
Seeds inside the capsule.
Propagation Through Seeds: The seeds should be collected during the month of
June to July after it gets ripened. Each berry of fruit of P. hexandrum contains small
dark brown seeds ranging between 30 and 69 depending upon the size of fruit and
locality. Seeds should be separated from pulp, washed under running tap water
for 20 min, and dried under shade and stored at 4
C until used. The seeds should
be sown in JuneeJuly immediately after collection. Seeds with hot water treatment
7.8 Good agricultural and collection practices 101
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for 24 h give a better germination percentage. They remain dormant for 9e
10 months and germination starts in following spring after the melt ing of snow.
Seed germination has been achieved with GA
conditions. In Hiko trays under polyhouse condition seed germination took place
within 55 days, while under nursery condition it took almost 210 days. In a field trial
it has been observed that potting mixture of humus:soil:sand in the ratio of 2:2:1
found to be good for germination percentage and growth. Transplantation of seedling in the field at a spacing of 30 30 cm should be carried out when seedlings
attain height of about 6e10 cm. About 4e5 Kg seeds per ha are required for direct
sowing (Sharma and Singh, 2014).
Seeds of P. hexandrum.
Vegetative Propagation: The multiplication of P. hexandrum is also done
through rhizome cuttings. The youngest top portion of the rhizome cuttings of
1.0e2.5 cm in length, bearing leaf primordium, leads to better sprouting in
P. hexandrum when planted in JuneeJuly in well-prepared soil at a spacing of
30e30 cm (Nautiyal and Nautiyal, 2003). Treatment of apical segments with
indole-3-butyric acid (IBA) or a-naphthalene acetic acid (NAA) increases rooting
percentage and also results in multiple root formation (Nadeem et al., 2000). GA
also shows marked effect in inducing uniform sprouting and flowering in rhizomes
of P. hexandrum grown at lower altitudes (Pandey et al., 2001).
200 ppm within 20 days under lab
3
3
Rhizome cuttings of P. hexandrum.
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Rhizome cuttings of 1.0e2.5 cm in length should be taken from the youngest tip
portion. The rhizome cuttings planted from May to the beginning of July gives the
best results. To improve vegetative multiplication, rhizome segments shoul d be
treated with NAA or IBA before planting; as rooting percentage was observed
almost double with IBA 100 ppm. The growth of plants is slow and it takes up 4e
5 years to produce rhizome suitable for exploitation (Sharma and Singh, 2014).
7.8.3 Transplantation and planting density
Under natural conditions P. hexandrum prefers organic mattererich light soil and
adequate moisture. The plant also prefers open slopes as well as partial shade condition for luxuriant growth (Singh et al., 2018). The rhizomatous system of the
P. hexandrum is deep and therefore soil must be at least 60 cm deep, free from
stones/pebbles, etc., for its optimum growth and good yield. Hence, these points
should be kept in mind before selection of cultivation site (Sharma and Singh,
2014). Due to slow growth of seedling, generally plants raised through vegetative
propagation are preferred as later takes less time for maturity. For the field
transplanting there should be 30 cm spacing between plant to plant and same distance for row to row. The planting materials, i.e., fresh rhizome of size 1e2cm
with growing apical buds, made from uprooted rhizome in MarcheApril to June
July should be planted 4e6 cm deep in the soil(Nautiyal and Nautiyal, 2003; Sharma
and Singh, 2014).
Transplanting of Podophyllum. hexandrum in field beds.
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7.8.4 Irrigation
Irrigation depends upon soil type and weather conditions. At the time of seedling
transplantation and plantation through rhizome cuttings, the crop requires watering
at alternate day during AprileMay. After proper establishment of plants, the crop
requires watering twice in a week. The crop undergoes dormant during frozen and
prolonged winters (NovembereMarch) and therefore there is no requirement of watering (Sharma and Singh, 2014).
7.8.5 Crop protection and maintenance
P. hexandrum is 3e6 years crop depending upon the mode of propagation followed.
Weeds growing along with crop affect the growth and yield of the plant. Hence, regular weeding is required for optimum growth and yield. Waterlogging conditions
should be avoided. In case of any insect pests attack, neem-based insecticide should
be used. However, no major insect pests have been observed under nursery and cultivation condition (Sharma and Singh, 2014).
7.8.6 Maturity of crop and harvesting
The crop grown through rhizomes becomes ready for harvest after 3 or 4 years of
plantation. However, crop raised from seed matures in 5e6 years after planting.
The rhizomes may be harvested at the time of senescence (OctobereNovember)
or at the time sprouting (AprileMay). The rhizomes harvested in spring (Aprile
May) are reported to contain higher resin content than those obtained in autumn
(OctobereNovember). Freshly harvested rhizomes are reported to contain higher
quantities of active ingredients (a.i.) and quantity of a.i. reduces after prolonged
storage.
7.8.7 Yield
The general cost norms for medicinal plants cultivation for the year 2017e18 as per
NMPB are as given below.
Sr. No. Expenses per acre Rupees
1. Cost norms (preparation of farm,
2. Other expenses 10,000.00
3. Harvesting, drying, and transport
hoeing and weeding, and irrigation)
58,564.00
5000.00
expenses
Total 73,564.00
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Sharma and Singh (2014) computed the economics of P. hexandrum. The details
are as given below.
1. Estimated production of dry roots
and rhizomes after 3 years(quintals/
acre)
2. Latest rates of market (Rs./kg) 450.00
3. Income from dried roots (Rs.) 90,000.00
4. The number of new plants produced
In the nursery from
seedsd20000 3 years
5. Income from selling plants (Rs. 5.0/
plant) in Rs.
6. Total income (Rs.) 1,20,000.00
7. Total expenses (Rs.) 73,564.00
8. Net profit after 3 years (Rs.) 46,436.00
2.0
60,000.00
30,000.00
7.8.8 Postharvest management
Fresh rhizomes should be immediately washed thoroughly with running water soon
after harvesting of the crop, so that all the foreign material is removed. For the ease
of drying the properly washed rhizomes are chopped with the help of sharp stainless
steel blade into small pieces of size 2e4 cm. Postharvest management prac tices are
very important to maintain the quality of crop. Rhizomes are the main parts which
contain the drugs and the trade depends upon the size and quality of rhizomes.
Hence, it is of paramount significance that scientific postharvest management practices should be followed to maintain the potency and vigor of drugs.
Drying and Storage: The choppe d rhizomes immediately need to be properly
dried. These fresh small pieces may be dried under partial shade which takes about
08e12 days for proper drying. Damp place for drying should be strictly avoided and
there should be proper ventilation, failing which results in fungal infestation of the
rhizomes, which will severely affect the quality of marketable product. The fresh
rhizomes can be dried in a dryer under hot circulating air at 45e50
20e24 h. The dried rhizome should have minimum 10%e12% moist moisture content. After drying the rhizomes are properly packed in gunny bags and kept in cool
places for storage. Storage in polythene bags should be avoided (Sharma and Singh,
2014).
C for about
7.9 Medicinal uses
It has been reported to be used through the ages and in modern times as a cure for
allergic and inflammatory conditions of the skin; biliary fever; burning sensation;
cold; constipation; cancer of the brain, bladder, and lung; erysipelas; Hodgkin’s
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