Pharmacognostic Studies on the Leaves of Hippophae rhamnoides L. and Hippophae salicifolia D. Don
N. Kasthuri Bai,
R. Mohan Kumar,
K. Ananth Kumar,
High altitude herbal medicines offers remedy for many diseases, particularly for which no medicine is available. As high altitude plants grow under stressful situations and exposed to high UV radiations, they are reported to have immense potential. Various scientific studies conducted on Hippophae species (Elaeagnaceae) during the last decade confirm its medicinal values. The present study was carried out with a view to lay down standards which could be useful to detect the authenticity of these medicinal plants, further the microscopical and physiochemical evaluation were studied to reveal the differences among Hippophae rhamnoides L. and Hippophae salicifolia D. Don. For the microscopic observation, free hand cross sections of the leaves of Hippophae rhamnoides and Hippophae salicifolia were stained with phloroglucinol and hydrochloric acid (1:1) and studied according to standard methods. Physiochemical analysis was carried out as per WHO guidelines on quality control methods for medicinal plants. The cross section of both the species of leaves were almost similar and revealed that the lower epidermal cells were completely covered with apressed stellate trichomes and abundant sunken stomata. Ash values of Hippophae rhamnoides leaves, showed higher ash content, compared to Hippophae salicifolia. The Pharmacognostic and phytochemical profile of Hippophae rhamnoides and Hippophae salicifolia are highly dependent on environmental adaptability of the plants. The present study helps in identification and differentiation of both the species of Hippophae.
to cite this article:
K. Ilango, N. Kasthuri Bai, R. Mohan Kumar, K. Ananth Kumar, G.P. Dubey and Aruna Agrawal, 2013. Pharmacognostic Studies on the Leaves of Hippophae rhamnoides L. and Hippophae salicifolia D. Don. Research Journal of Medicinal Plants, 7: 58-67.
Received: December 06, 2012;
Accepted: February 13, 2013;
Published: April 18, 2013
Plants grown in the Himalayan region is a rich repository of medicinal wealth,
occupying an important place in vedic treatise, due to its wide range
of altitudes, topography and climatic conditions, medicinal species found in
this part of India is more commonly used by the local communities, since time
immemorial for curing various ailments of human kind (Li
et al., 2006). Leaf morphological and physiological characteristics
are extremely variable across environmental gradients in most of the woody plant
due to their adaptable nature. The pharmacognostical study of the plant material
is mainly concerned with the description, identification including history of
use, commerce, collection, preparation and mainly for quality control purpose.
To ensure reproducibility and quality of plant based drugs, authentication is
essential (WHO, 1992). The present pharmacognostic study
on two Seabuckthorn species provides the preliminary pharmacognostic profile
and standards of these plants which has shown multi-dimensional pharmacological
activities. According to World Health Organization, the macroscopical and microscopical
description of medicinal plant is the main parameter for establishing the identity
and purity assessment of medicinal plants.
Hippophae genus (Elaeagnaceae) consists of five species, based on morphological
variations viz., H. rhamnoides L., H. salicifolia D. Don, H.
neurocarpa Liu and He, H. tibetana Schlecht and H. goniocarpa.
The main species of Seabuckthorn distributed in India are H. rhamnoides,
H. salicifolia and H. tibetana. Seabuckthorn is a naturally growing
thorny, deciduous bush, indigenous to the Himachal Pradesh, is fast emerging
as an important crop due to its nutritional and medicinal properties. With the
commercialization of H. rhamnoides products, demand of raw material has
increased manifold (Ting et al., 2011). In India
it grows mainly at high altitude, cold arid conditions of Ladakh, Himachal Pradesh.
H. rhamnoides possess characteristic features like terminal and lateral
thorns. The thorn per 10 cm was observed 4-6 per 10 cm in young plants. The
plants rapidly spread by rhizomatous roots and suckers which quickly spread
and colonize in the adjacent areas. The root is light-colored and thick (Kritikar
and Basu, 2003).
Various scientific studies conducted on H. rhamnoides during the last
decade confirm its medicinal and nutritional values. The important phytochemicals
present in this plant is flavonoids, carotenoids, fatty acids etc., showing
various biological activities (Li et al., 2006).
More than 100 different kinds of phyto-nutrients and bioactive substances are
present in the leaves and berries including vitamins, fatty acids, free amino
acids and minerals. The vitamin-C content is 5-10 times higher than any other
known fruit or vegetable (Zu et al., 2006). Hippophae
possesses common know biological activities like antitussive, carminative (Li
and Jie, 2010). Leaves are used in gastro intestinal and dermatological
disorders and have been applied as compress form in rheumatoid arthritis (Suryakumar
and Gupta, 2011).
The plant Seabuckthorn has shown multi-dimensional therapeutic activity including
immuno-modulatory, neuro-modulatory, anti-oxidant, anti-inflammatory and anti-stress
roles (Dubey et al., 2002). The immuno-modulatory
property of H. rhamnoides is well established in several studies. The
ripe fruits of H. rhamnoides contain malic acid, oxalic acid, phospholipids
and vitamins like A, B complex, C, E and K. Other nutrients like fat, protein,
organic acids and flavonoides are also found (Gupta et
al., 1990). Several recent studies showed that H. rhamnoides contains
biologically active substances which can enhance the immunity and reduces the
cardiovascular disorder which may be due to the presence of 5-HT in the peel
of stem and fruits which is rare occurrence in plant kingdom (Dubey
et al., 1990). The Phytoconstituents extracted from the H. rhamnoides
strengthens nonspecific immunity (Agrawal and Goel, 2002;
Goel et al., 2005). The plant also showed a
potent effect on age related deterioration of cognitive functions (Agrawal
et al., 2000).
Thirty five hypertensive patients received the Hippophae extract for
eight weeks, the total flavones present in the H. rhamnoides, prevented
supine exercise induced increase in heart rate, blood pressure and plasma catecholamine
concentration (Zhang et al., 2001). H. rhamnoides
oil also possesses hepato-protective activity (Gao et
al., 2003). Sea buckthorn leaves can be used as food additives and can
be utilized for the development of useful natural compounds, since they show
antioxidant and α-glycosidase activity (Kim et
al., 2011). The aqueous leaf extract has shown significant healing property
in burn wounds and has a beneficial effect on the different phases of wound
repair and also it has anti-depressant activity (Upadhyay
et al., 2011; Batool et al., 2011).
Thus, this plant has a wide therapeutic application in the prevention and management
of various diseases.
H. salicifolia is restricted to Himalayan region at an altitude of 1500-3500
m. They are reported to be one of the best species of the genus Hippophae,
since they yield a high quality fruit. The plant can withstand temperatures
from -43-40°C, grows in areas with mean annual temperatures ranging from
4.7-15.6°C and with annual precipitation ranging from 250-800 mm. Hippophae
plants are highly adaptable to various soil types. They can grow in hill and
gully tops, where the water content is very low (only 15%). They can also survive
in valley or gully bottoms with 1.15 salts content. H. salicifolia is
deciduous shrub or a small tree with thorns. The plant bears foliage from April
to November, flowers during June-July for a week and fruits formed during mid
August to April. Female plants of H. salicifolia bear red, yellow or
orange coloured fruits which is usually 1 cm across (Basistha
et al., 2010).
All the species and subspecies of Seabuckthorn possess similar morphological
characters (Guofu et al., 2006) which makes
the identity and collection of the plants difficult. The review of literature
revealed that, no pharmacognostic studies have been conducted on this plant.
Hence the present pharmacognostic studies of two species i.e., H. rhamnoides
and H. salicifolia D. Don has been undertaken with the objective to establish
pharmacognostic and phytochemical standardization of the leaves, so that authentic
plant material could be explored for its therapeutic claims.
MATERIALS AND METHODS
Collection of plant material: The plant material proposed for the study
was collected in the month of September 2011 from Ladak district of Kashmir
by Dr. D.P. Attrey, Former Director, Defense Institute of High Altitude Research
[formerly Field Research Laboratory (FRL)], Leh, India. The plant specimen was
identified by Prof. N.K. Dubey, Professor in Plant Taxonomy, Department of Botany,
Faculty of Science, Banaras Hindu University, Varanasi, India. A voucher specimen
(HR0706/SH18 and HR0707/SH19) has been preserved in the laboratory for future
Instruments used: Photographs of different magnifications were taken with a Leica-camera inbuilt, inverted binocular microscope. Bright field was used for observations.
Macroscopic and microscopic studies: The gross morphological character was
described based on the shape, size, colour and surface of the leaves. For the
microscopic observation, transverse sections of leaves were stained with phloroglucinol
and hydrochloric acid, observed through microscope and were studied following
standard methods (O'Brian et al., 1964; Esau,
1964). Coarse powder of mesh size 60 was cleared with sodium hydroxide and
mounted in glycerin medium after staining with phloroglucinol and hydrochloric
acid to study the powder characteristics of the leaf.
Physiochemical analysis: The dried powdered leaf material was subjected
to physicochemical analysis including fluorescence analysis (Kokoski
et al., 1958), moisture content, total ash, water soluble ash, acid
insoluble ash and extractive values to determine the quality and purity of the
plant materials (WHO, 1992).
Preliminary phytochemical screening: The leaves were shade-dried, coarsely
powdered with a mechanical grinder and passed through a 40-mesh sieve. The sieved
powder material was stored in an air-tight container and kept at room temperature
till further study. The dried powder material was extracted with hexane, chloroform,
ethyl acetate, methanol and water by cold maceration. The solvents were completely
removed under reduced pressure using vacuum evaporator. The presence of various
phytoconstituents like, alkaloids (Dragendorffs test), steroids and terpenoids
(Libermann- Burchard test), tannin and phenolic compounds (Ferric chloride test),
flavonoids (Shinoda test), amino acids (Ninhydrin test) etc., were detected
following methods developed by (Kokate, 1986; Harborne,
Hippophae rhamnoides: Leaves alternate; petiolate; leaf blade abaxially
silvery white suffused with brown or yellow, adaxially dark greyish green, linear
or linear-lanceolate, 2-8x0.2-0.8 cm, narrowed at base, abaxially with white
and brown stellate scales, margin revolute, apex sub obtuse (Fig.
Hippophae salicifolia: Leaves blade abaxially whitish with usually reddish brown midrib, adaxially green, linear-oblong, 4.2-6.2x0.6-1.2 cm, abaxially tomentose, adaxially stellate-hairy, margin usually revolute (Fig. 1).
Anatomical characters: The cross sections of both species leaves were
almost similar and revealed the presence of same type of cells. They are dorsiventral
with two layers of palisade cells below the upper epidermis, the palisade cells
are absent in the mid-rib region. The transverse section of the leaves of H.
rhamnoides and H. salicifolia showed a layer of upper and lower epidermis
covered with thin cuticle. The epidermal cells of the adaxial surface are slightly
bigger in size than the abaxial region.
|| Macroscopy of the dried leaves (a) Hippophae rhamnoides
and (b) Hippophae salicifolia
The lower epidermal cells were completely covered with pressed stellate trichomes.
Both H. rhamnoides and H. salicifolia leaves had sunken stomata
in both the epidermis. The silvery appearance of the leaf at the ventral surface
is due to the dense covering of the trichomes which is the characteristic identity
of the Hippophae leaves. The midrib portion of the leaf composed of 5-6
layers of collenchyma cells below the upper epidermis which is followed by xylem
vessels which stained pink with phloroglucinol-HCl, due to the presence of lignin.
Three to four layers of phloem cells are found beneath the xylem vessels and
Six to seven layers of spongy parenchyma is seen above the lower epidermis (Fig.
2). Small difference was observed in the shape of the midrib. H. rhamnoides
showed a heart shaped midrib, whereas the shape of the midrib of H. salicifolia
is concave. Lower epidermis of the lamina show densely covered trichomes with
sunken stomata, vascular strands and loosely packed spongy parenchyma.
Powder microscopy of two species leaves revealed the presence of different
types of trichomes (Fig. 3). stellate, peltate and a combination
of both stellate-peltate trichomes, broken fragments of epidermis with palisade
cells and broken xylem vessels.
||Transverse section of the leaf through the midrib and lamina
(a) Transverse section of the leaf through the midrib and (b) Transverse
section of the leaf through the lamina, EP: Epidermis, CO: Collenchyma,
TR: Trichome, HD: Hypodermis, VB: Vascular bundle, LV: Lateral vein, VS:
Vascular strand, SP: Spongy parenchyma, SS: Sunken stomata and CU: Cuticle
||Different types of Trichomes observed in powder microscopy
of the leaf (a) H. rhamnoides and (b) H. salicifolia
|| Preliminary phytochemical analysis of H. rhamnoides
and H. salicifolia
|+: Present, -: Absent
Preliminary Phytochemical screening: Preliminary phytochemical screening of various extracts of the leaves of Hippophae revealed the presence of steroids, terpenoids, saponins, flavonoids, phenolic compounds and carbohydrates (Table 1).
Physicochemical analysis: The physicochemical parameters such as ash
values, loss on drying (moisture content), extractive values, fluorescence analysis
and foaming index were measured and are depicted in Table 2.
The total ash value of H. rhamnoides (80) is less when compared to H.salicifolia
(85.60). The leaves of H. salicifolia contain less of water soluble ash
(4.09) when compared to H. rhamnoides (10). Water soluble ash signifies
the physiological content of the leaf. The results of extractive values are
shown in Table 3. Powder characteristics of the leaves when
treated with various chemicals were observed under the UV and day light, explores
the presence of nature of Phytoconstituents present in the leaves.
|| Extractive values of the leaves of H. rhamnoides and
|| Physiochemical characters
|| Fluorescence analysis of Hippophae sp. leaf powder
Green fluorescence was observed under the UV light, when the leaf powder was
treated with NaOH signifying the presence of flavonoids (Table
A major drawback of herbal medicine is lack of standardization and quality
control. The main importance is with respect to quality control for correct
identification of the species concerned, whether in the fresh, dried or powdered
state (Springfield et al., 2005). The misclassification
of species and the mistaken substitution is a real danger in the preparation
and administration of herbal medicine (Elizabeth, 2004).
In the present study microscopical evaluation and physiochemical analysis of
two Hippophae species were carried out. Microscopic evaluation of H.
rhamnoides and H. salicifolia leaves revealed that, they look similar
in the type of cells except slight difference like shape of the midrib and the
presence of hypodermis. Powder microscopy of the leaves showed the presence
of different types of trichomes which is characteristic identity of the Hippophae
species. The dense covering of stellate trichomes can be a good cover of shelter
in preventing the high temperature of the leaves, sunken stomata and the dense
epidermal cells cover; can effectively reduce the water loss showing the plant
to be high drought resistant property. The characters available in the powder
are much fewer than the potentially available characters in whole specimens.
The difference is attributable to the damage of the plant cell wall during preparation,
causing distortion in tissues arrangements and patterns normally found in the
untreated plant samples. The characters available in the powder form of the
specimens are potentially useful for distinguishing the samples even in mixture
(Jayeola, 2009). The powder microscopy of both the
species revealed the presence of three different types of trichomes, stellate,
peltate and combination of both the types- Stellate-peltate trichomes which
are characteristic feature of the Hippophae sp. Preliminary phytochemical
analysis of all two species of Seabuckthorn indicated the presence of steroids,
terpenoids, flavonoids, coumarin glycosides, phenolic compounds and saponins.
The analysis of ash values revealed that, the total ash, acid insoluble ash
and water-soluble ash are present in different quantities in both the species.
The ash content of H. salicifolia (85.60% w/w) is higher than H. rhamnoides
(80% w/w). The extractive values are useful to evaluate the chemical constituents
present in the crude drug and also helpful in estimation of specific soluble
constituents in a particular solvent. Extractive values of the leaves of two
species showed that the methanolic and water extractive values were more when
compared with other solvents.
The present study was undertaken with a view to lay down standards which could be useful to detect the authenticity of these medicinal plants. Microscopic study and physicochemical standards can be useful to substantiate and authenticate the drug. Seabuckthorn, an underutilized and neglected plant of the cold arid region is a goldmine with an untapped potential. Efforts towards preparation of food products from Seabuckthorn have suitably highlighted its immense potential commercially.
The pharmacognostic and the phytochemical profile of H. rhamnoides and H. salicifolia are highly dependent on environmental adaptability of the plants. The present study is helpful in identification of the H. rhamnoides, H. salicifolia and various sub species of this plant.
The authors would like to thank the Department of Science and Technology, Government of India, for providing the financial assistance to carry out the work.
Jayeola, A.A., 2009.
Micro morphological study of plant fragments in some powdered medicinal plants. J. Med. Plants Res., 3: 438-442.Direct Link |
Agrawal, A., S.P. Dixit and G.P. Dubey, 2000.
Role of herbal formulation in the management of age related neuro psycho physiological deterioration among the elderly. Ind. J. Gerontol., 14: 15-23.
Agrawala, P.K. and H.C. Goel, 2002.
Protective effect of RH-3 with special reference to radiation induced micronuclei in mouse bone marrow. Indian J. Exp. Biol., 40: 525-530.PubMed |
Quality Control Methods for Medicinal Plant Materials. World Health Organisation, Geneva, Switzerland
Basistha, B.C., N.P. Sharma, L. Lepcha, M.L. Arrawatia and A. Sen, 2010.
Ecology of Hippophae salicifolia
D. Don of temperate and sub-alpine forests of North Sikkim Himalayas-a case study. Symbiosis, 50: 87-95.CrossRef | Direct Link |
Li, C., X.U. Gang, R. Zang, H. Korpelainen and F. Berninger, 2006.
Sex-related differences in leaf morphological and physiological responses in Hippophae rhamnoides
along an altitudinal gradient. Tree Physiol., 27: 399-406.Direct Link |
Dubey, G.P., A. Agrawal, B.S. Gupta and K.N. Udupa, 1990.
Neurophysiological adaptation following cold stress under the influence of Badriphal (Hippophae rhamnoides
L.). Pharmacopsychoecologia, 3: 59-63.
Dubey, G.P., A. Agrawal and S.P. Dixit, 2002.
Role of Seabuckthorn (Hippophae rhamnoides
) in the maintenance of cardiovascular homeostasis following cold stress. J. Nat. Rem., 3: 136-140.Direct Link |
Esau, K., 1964.
Plant Anatomy. 2nd Edn., Wiley and Sons, New York, Pages: 767
Batool, F., A. Kamal, M. Sattar, A.H. Shah, S.D. Ahmed, Z.S. Saify and D.J. Haleem, 2011.
Evaluation of antidepressant-like effects of aqueous extract of sea in experimental models of depression. Pak. J. Bot., 43: 1595-1599.Direct Link |
Li, G. and Y. Jie, 2010.
[Leaf morphological structure and physiological and biochemical characteristics of female and male Hippophae rhamnoides subsp. sinensis under different soil moisture condition]. Ying Yong Sheng Tai Xue Bao., 21: 2201-2208.PubMed | Direct Link |
Gao, Z.L., X.H. Gu, F.T. Cheng, F.H. Jiang, 2003.
Effect of sea buckthorn on liver fibrosis: A clinical study. World J. Gastroenterol., 9: 1615-1617.PubMed |
Suryakumar, G. and A. Gupta, 2011.
Medicinal and therapeutic potential of Sea buckthorn (Hippophae rhamnoides
L.). J. Ethnopharmacol., 138: 268-278.CrossRef | Direct Link |
Goel, H.C., D. Gupta, S. Gupta, A.P. Garg and M. Bala, 2005.
Protection of mitochondrial system by Hippophae rhamnoides
L. against radiation-induced oxidative damage in mice. J. Pharm. Pharmacol., 57: 135-143.PubMed | Direct Link |
Gupta, B.S., A. Agrawal, G.P. Dubey and K.N. Udupa, 1990.
Anti-ageing effects of Badrifal (Hippophae rhamnoides
L). Pharmacopsychoecologia, 3: 53-57.
Harborne, J.B., 2005.
Phytochemical Methods-A Guide to Modern Techniques of Plant Analysis. 3rd Edn., Springer Pvt. Ltd., New Delhi
Ting, H.C., Y.W. Hsu, C.F. Tsai, F.J. Lu, M.C. Chou and W.K. Chen, 2011.
The in vitro
and in vivo
antioxidant properties of seabuckthorn (Hippophae rhamnoides
L.) seed oil. Food Chem., 125: 652-659.CrossRef | Direct Link |
Kim, J.S., Y.S. Kwon, Y.J. Sa and M.J. Kim, 2011.
Isolation and identification of Seabuckthorn (Hippophae rhamnoides
) phenolics with antioxidant activity and α-glucosidase inhibitory effect. J. Agric. Food Chem., 59: 138-144.CrossRef | PubMed | Direct Link |
Kokoski, C.J., R.J. Kokoski and F.J. Slama, 1958.
Fluorescence of powdered vegetable drugs under ultraviolet radiation. J. Am. Pharmaceut. Assoc., 47: 715-717.CrossRef | Direct Link |
Kokate, C.K., 1986.
Practical Pharmacognosy. 4th Edn., Vallabh Prakashan, Delhi, Pages: 146
Kritikar, K.R. and B.D. Basu, 2003.
Indian Medicinal Plants. Vol. 3, Oriental Enterprises, India, pp: 75-77
Upadhyay, N.K., R. Kumar, M.S. Siddiqui and A. Gupta, 2011.
Mechanism of wound-healing activity of Hippophae rhamnoides
L. leaf extract in experimental burns. Evi. Based Complement Alternat Med., Vol. 2011.CrossRef |
O'Brien, T.P., N. Feder and M.E. McCully, 1964.
Polychromatic staining of plant cell walls by toluidine blue O. Protoplasma, 59: 368-373.CrossRef | Direct Link |
Elizabeth, O.L., 2004.
The efficacy and safety of Chinese herbal medicines. Br. J. Nut., 91: 171-193.CrossRef | Direct Link |
Springfield, E.P., P.K. Eagles and G. Scott, 2005.
Quality assessment of South African herbal medicnes by means of HPLC fingerprinting. J. J Ethnopharmacol., 101: 75-83.CrossRef | PubMed | Direct Link |
Guofu, W., L. Lianguo, L. Xiaoyan, D. Shiyong, Z. Liying and X. Lifen, 2006.
Relationship between the morphological characteristics of leaf surface and drought resistance of Seabuckthorn. Acta Hortic. Sinica, 33: 1310-1312.Direct Link |
Zu, Y., C. Li, Y. Fu and C. Zhao, 2006.
Simultaneous determination of catechin, rutin, quercetin kaempferol and isorhamnetin in the extract of sea buckthorn (Hippophae rhamnoides
L.) leaves by RP-HPLC with DAD. J. Pharm. Biomed. Anal., 41: 714-719.CrossRef | PubMed |
Zhang, X., M. Zhang, Z. Gao, J. Wang and Z. Wang, 2001.
Effect of total flavones of Hippophae rhamnoides
L. on sympathetic activity in hypertension. J. West China Univ. Med. Sci., 32: 547-550.Direct Link |