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 Table of Contents  
ORIGINAL ARTICLE
Year : 2015  |  Volume : 9  |  Issue : 2  |  Page : 102-108

Pharmacognostic standardization of Homoeopathic drug: Juniperus virginiana L.


1 Research Officer, Scientist-4, Drug Standardisation Unit, Habsiguda, Hyderabad, Telangana, India
2 Pharmacognosy, Chemistry, Drug Standardisation Unit, Habsiguda, Hyderabad, Telangana, India
3 Senior Research Fellow (Botony), Drug Standardisation Unit, Habsiguda, Hyderabad, Telangana, India
4 Department of Botony Osmania University, Hyderabad, Telangana, India

Date of Submission09-Oct-2014
Date of Acceptance12-Jun-2015
Date of Web Publication30-Jun-2015

Correspondence Address:
Dr. P Subramanian
Research Officer, Scientist 4 (Chemistry), Drug Standardisation Unit, O.U.B. 32, Road No. 4, Habsiguda, Hyderabad - 500 007, Telangana
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0974-7168.159539

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  Abstract 

Background: Juniperus virginiana L., commonly known as 'red cedar' in English is a well-known evergreen tree belonging to the family Cupressaceae. The leaves and young aerial shoots are used for preparation of medicine in Homoeopathy.
Objective: Standardization is the quintessential aspect which ensures purity and quality of drugs. Hence, the pharmacognostic and physico-chemical studies are carried out to facilitate the use of authentic and correct species of raw drug plant material with established parametric standards for manufacturing the drug.
Materials and Methods: Pharmacognostic studies on leaves and young aerial parts of authentic samples of J. virginiana L. have been carried out; physico-chemical parameters of raw drug viz., extractive values, ash values, formulation, besides weight per mL, total solids, alcohol content along with High Performance Thin Layer Chromatography (HPTLC) and ultraviolet visible studies have been worked out for mother tincture.
Results: The leaves are needles, narrow and sharp at tips; stems are round with greyish white to brown bark possessing small lenticels and covered by imbricate leaves. Epidermal cells in the surface have polygonal linear sides with pitted walls containing crystals and starch. Stomata exclusively occur on the adaxial surface in linear rows. Hypodermis of leaf in T.S. is marked with 1-2 layered lignified sclerenchyma. 2-4 secretory canals are present with one conspicuously beneath midvein bundle. The young terminal axis is sheathed by two closely surrounding leaves while the mature stem possess four leaf bases attached. Vascular tissue of stem possesses predominant xylem surrounded by phloem containing sphaeraphides, prismatic crystals and starch grains. Uniseriate rays occur in the xylem. Mature stem possess shrivelled cork, followed by the cortex. Physicochemical properties and HPTLC values of the drug are standardized and presented.
Conclusion: The powder microscopic features and organoleptic characters along with anatomical and physicochemical studies are diagnostic to establish the standards for the drug.

Keywords: High performance thin layer chromatography, Juniperus virginiana, Pharmacognosy, Secretory canal, Standardization


How to cite this article:
Rao P P, Subramanian P, Sudhakar P, Reddy P R. Pharmacognostic standardization of Homoeopathic drug: Juniperus virginiana L. Indian J Res Homoeopathy 2015;9:102-8

How to cite this URL:
Rao P P, Subramanian P, Sudhakar P, Reddy P R. Pharmacognostic standardization of Homoeopathic drug: Juniperus virginiana L. Indian J Res Homoeopathy [serial online] 2015 [cited 2018 Jan 16];9:102-8. Available from: http://www.ijrh.org/text.asp?2015/9/2/102/159539


  Introduction Top


Juniperus virginiana L., commonly known as 'red cedar' in English is an evergreen tree belonging to family Cupressaceae. It is grown from Canada to Gulf of Mexico, Westward to Texas and Neveda to British Columbia. [1] It is also found in Nilgiris, India. Leaves and young aerial parts are used for preparation of medicine in Homoeopathy. Medicinally, it is used in apoplexy, convulsions, strangury, tetanus, uterine haemorrhages and affection of eyes. [2],[3] Its history and authority about the proving of drug is mentioned in Homœopathic Pharmacopœia of the United States [HPUS]. [4] Chemically, the plant is reported to contain volatile oils, cedrenol, pseudocedrenol, cedrene isomers, bicyclic sesquiterpenes, podophyllotoxin, oleoresins, cedrol [Figure 1], thujopsene and juniperin. [5],[6],[7],[8]
Figure 1: Chemical structure of cedrol

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The pharmacognostic studies on leaves and young shoots were not conducted yet. A part from histo anatomical researches in Cupressaceae, Considering the medicinal importance of the drug in Homoeopathy and absence of earlier pharmacognostic studies, this study is undertaken.


  Materials and Methods Top


Pharmacognosy

The plant material J. virginiana was supplied by the Survey of Medicinal Plants and Collection Unit, Nilgiris, Tamil Nadu. The leaves and stems were fixed in formaldehyde acetic acid alcohol, dehydrated through xylene alcohol series, embedded in paraffin wax. The cross sections between 8 - 10 μm were stained in crystal violet and basic fuchsin combination as per the Johansen method. [9] Epidermal peels were obtained by gently scraping and peeling by the razor blade. Then peels were stained in saffranine and mounted in glycerine. The photomicrography was done on Olympus BX-53 trinocular microscope (Japan) attached with digital Sony Camera.

Physicochemical

Air-dried leaves and stems were coarsely powdered to 10/44 (sieve size) and subjected to determination of loss on drying at 105°C, total ash, water soluble ash, acid insoluble ash, extractability in different solvents, physicochemical constants, ultraviolet (UV) aspects of mother tincture following official methods. [10] Mother tincture was prepared as per Homoeopathic Pharmacopoeia of India [HPI] by percolation method. [10]

High-Performance Thin Layer Chromatography Analysis

25 mL mother tincture was evaporated on a water bath to remove the alcohol. The residue was extracted thrice with 25 mL chloroform. The concentrated chloroform extract was used for the High Performance Thin Layer Chromatography (HPTLC) study. The concentrated chloroform extract was spotted in the form of band of width 4 mm with a Camag microliter syringe on precoated Silica gel aluminum plate 60F-254, (5 cm × 10 cm with 0.25 mm thickness; Merck, Darmstadt, Germany) using a Linomat IV sample applicator (Camag, Muttenz, Switzerland, supplied by Anchrom technologists, Mumbai). A constant application rate of 6 μL/s was employed. The slit dimension was kept at 4 mm × 0.45 mm and 20 mm/s scanning speed was employed. The mobile phase consisted of Chloroform: Methanol (9:1 v/v) and 10 mL of mobile phase was used for Chromatography. Linear ascending development was carried out in a 10 cm × 10 cm twin trough glass chamber (Camag, Muttenz, Switzerland) saturated with the mobile phase at room temperature for 20 minutes. The length of the chromatogram run was 8 cm and subsequent to the development, the TLC plates were dried in a current of air with the help of hot air dryer in a wooden chamber with adequate ventilation. Densitometric scanning was performed (Camag TLC scanner III- Camag Switzerland) at 254 nm and 366 nm by reflectance scanning and operated by Wincats software (Camag) resident in the system. [11],[12],[13]


  Observations and Results Top


Morphology

Evergreen tree upto 30 meter high; branches horizontal and surface of covered with minute knots; twigs covered with densely imbricated leaves, which increase in size and become broken and confounded with the bark; tree yields small bluish berries, male cones small, greenish yellow.

Leaf

Macroscopy


Leaves are green, needles sessile, 0.8-1.2 cm long and 1-1.5 mm wide, narrow, tip sharp, surface adaxially reticulate and abaxially ridged with a median simple vein.

Microscopy

Leaf - surface


Epidermal cells 5-6 sided, polygonal linear, sides slightly thick, straight to curved and few wavy on abaxial, often pitted; surface smooth on adaxial and striated on abaxial; contents dense with crystals of Calcium oxalate and starch grains in few. Distributed: Irregularly and, parallelly oriented [Figure 2]b.

Stomata restricted to adaxial, anomocytic, subsidiaries 5 or 6, indistinct, guard cells linear reniform, contents dense, ledged. Distributed: in linear patches of 2-3 rows, parallelly oriented. Stomatal sizes 44-80 μm (58) long and 16-27 μm (22) wide [Figure 2]a. Uniseriate peltate scaly hair, few restricted to lower half of leaf on abaxial.
Figure 2: (a) Leaf adaxial surface x 111, (b) Leaf abaxial surface x 486, (c) T.S. of leaf x 132, (d) T.S. of young stem axis x 149. pe: Pitted epidermis, s: Stomata, st: Starch grains, sph: Sphaeraphides, e: Epidermis, l: Lignified hypodermis, sc: Secretory canal, v: Vascular bundle, m: Mesophyll, c: Cortex, ph: Phloem, x: Xylem

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In T.S. the leaf is elliptic in outline, ribbed on either sides at midvein, margins obtuse; midvein 302-368 μm (336) and lamina 150-260 (198) μm thick. Epidermis is one layered, with few sunken stomata on adaxial side. Cells oval to spherical, oblong and barrel-shaped, walls slightly thick, contents scanty and in few dense with sphaeraphides of Calcium oxalate, covered by a thick cuticle. 1-2 layered stone cells are characteristic and occur as hypodermis. Mesophyll is 3 to 5 layered made of polygonal and elongated parenchyma cells towards adaxial containing chloroplasts and sphaeraphidal crystals. Cells in middle and abaxial sides are polygonal or rounded in often containing prismatic crystals and chloroplasts with large intercellular spaces. The mesophyll also possess 2-4 oil secretory canals, one conspicuously beneath the central vascular bundle [Figure 2]c.

Vascular tissue consists of a central spherical vascular bundle 202-280 μm (220) in diameter, endarch, collateral with a sclerenchymatous layer enclosing the phloem. A layer of endodermis encloses the vascular bundle [Figure 2]c.

Young Stem

Macroscopy


Rounded, up to 3 mm thick, surface, greyish to brown, rough, with four leaf bases fused at corners appearing quadrangular, internally creamish.

Microscopy

The epidermis at leaf base is one layered covered by a thick cuticle. Hypodermis consists of a layer of sclereidal fibers, which are radially long. The mesophyll is 8-10 layered with some peripheral cells filled with sphaeraphidal crystals and a few with chloroplasts and starch grains. A spherical secretory canal occurs at the periphery in the hypodermal area enclosed by a 2-3 layered epithelium. The inter angled furrowed region has single layered epidermis with a thick cuticle. A 6 to 8 layered cortex with radially elongated cells is present. Secondary phloem consists of phloem parenchyma, sclereidal fibers, sieve cells and medullary rays. Phloem parenchyma cells possess sphaeraphides, prismatic crystals and starch grains. Sclereidal fibers occur in transverse bands. Medullary rays are uniseriate often containing crystals [Figure 3]a.
Figure 3: (a) T.S. of young stem x 62, (b) T.S. of mature stem x 185, (c) T.S. of mature stem with crystals × 158. e: Epidermis, l: Lignified hypodermis, m: Mesophyll, sc: Secretory canal, c: Cortex, ph: Phloem, x: Xylem, r: Xylem rays, pi: Pith, cr: Crystals, ck: Cork

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Centrally the wood is abundant with tracheids and medullary rays. Tracheids are rectangular to tabular arranged in radial rows interrupted by uniseriate rays, which are 2-6 cells high often containing starch grains. The tracheids are long with circular bordered or field pits arranged in single rows. Centrally the pith is scanty, parenchymatous and appears radiating in 3-5 sides. Cells are polygonal, slightly larger with dense contents [Figure 3]a.

T.S. mature stem

The stem is spherical in outline covered with remnants of the leaf. The structure of leaf is similar as described in the young stem. Few branch traces occur with precocious cortex. The stem consists of outermost shrivelled cork followed by the cortex which is scanty and compressed. The cortex possesses few crystalliferous cells. Secondary xylem or wood is as described in the young stem but more extensive. Centrally pith is scanty [Figure 3]b and c.

Terminal Axis

Macroscopy


Slender, oblong in T.S. upto 1 mm thick, sheathed by young leaves, green.

Microscopy

In T.S. oblong, enclosed by two young leaves, which appear merged with the stem. The leaf mesophyll possesses sphaeraphides and prismatic crystals in few. Stem thin 216-302 μm (244) in diameter. Epidermis is one layered and followed by 3 to 4 layered cortex made of polygonal to elongated cells in tangential bands enclosing vascular tissue. The vascular tissue is surrounded by phloem which possess crystalliferous idioblasts [Figure 2]d.

Physico-chemical Studies

The determined data under the physico-chemical study for the raw drug is summarized in [Table 1] and that of mother tincture preparation and its standardization in [Table 2] and [Table 3] respectively.
Table 1: Standardization of raw drug


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Table 2: Formulation of mother tincture (percolation technique used)


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Table 3: Standardization of mother tincture


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Qualitative Phytochemical Tests

Loss on drying reveals the presence of water in the plant and also some volatile organic matter. Results of physico-chemical studies are summarized in [Table 1], [Table 2], [Table 3].

High Performance Thin Layer Chromatography Fingerprinting

The profile of chromatographic separation scanned at 254 nm, reveals seven spots [Figure 4] and [Figure 5] out of which 1, 2, 5 and 7 spots possess maximum composition with Rf at 0.24, 0.36, 0.63 and 0.80 respectively. While, chromatogram scanned at 366 nm, revealed 9 spots with 6, 7, 8 and 9 spots showing the maximum composition at Rf 0.42, 0.51, 0.68 and 0.83 respectively. It is evident from the data that these are characteristic for the studied drug, which will help in identification and authentication of the mother tincture. These are considered valuable standards in Pharmacopoeia. At 254 nm, seven spots appear at Rf 0.24 (dark brown), 0.36 (dark brown), 0.45, 0.51 (light brown), 0.63 (dark brown), 0.74 (light brown) and 0.80 (dark brown) [Figure 6] with various concentrations while at 366 nm, nine spots appears at Rf , 0.17, 0.23, 0.32 (light red), 0.42 (red), 0.45 (light red), 0.51 (blue), 0.68, 0.81 and 0.83 (red). These are a vital finger print parameters to ensure the reliability and reproducibility of the drug.
Figure 4: High Performance Thin Layer Chromatography finger printing (chloroform: Methanol 9:1 v/v) of Juniperus virginiana Φ scanned at 254 nm

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Figure 5: High Performance Thin Layer Chromatography finger printing (chloroform: Methanol 9:1 v/v) of Juniperus virginiana Φ scanned at 254 nm

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Figure 6: High Performance Thin Layer Chromatography finger printing (chloroform: Methanol 9:1 v/v) of Juniperus virginiana Φ scanned at 366 nm

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  Discussion Top


Pharmacognosy

Juniperus virginiana L. is a large evergreen tree with horizontal branches. The leaves are green, sessile needles. The young stem is greyish, slender with minute knots and covered with leaf bases and appear quadrangular. The epidermal cells in the surface are polygonal linear with sides straight to curved and few wavy on abaxial and parallelly oriented. Leaves are epistomatic with stomata in 2-3 rows and anomocytic as also reported earlier. Few, uniseriate peltate scaly hair are present on leaf abaxial. 1-2 layered lignified hypodermis made of stone cells is characteristic as also reported. [14] The assimilatory parenchyma was reported as reversed heterofacial bifacial in J. virginiana[14] but presently it is undifferentiated.

The 2 to 4 secretory ducts are present in the leaf with one conspicuously beneath the central vascular bundle close to endodermis in leaf and confirms earlier studies. [14] The presence of idioblasts with sphaeraphides and prismatic crystals in mesophyll is characteristic.

A spherical secretory canal occurs in hypodermal areas at four corners of the stem. Phloem parenchyma possess idioblasts with starch grains, sphaeraphides and prismatic crystals. Centrally the secondary xylem is abundant interrupted by unilayered medullary rays, which contain starch grains. Pith is scanty and appears stellate radiating on 3-5 sides. The mature stem has shrivelled cork followed by cortex toward the exterior. Secondary phloem is extensive interrupted by medullary rays. Secondary xylem is also extensive, and pith is reduced. The T.S. terminal axis (stem) is slender, enclosed by the leaves, which appear merged. The vascular tissue is enclosed by phloem containing crystalliferous idioblasts.

Physicochemical

The phytochemical analysis using various reagents showed the presence of secondary metabolites like tannins and phenolic compounds, alkaloids, and volatile oils. Physico-chemical constants viz., ash and extractive values can be used as a reliable aid to check the identity, purity and strength.

Thin Layer Chromatography is done as an important tool for the qualitative and quantitative analysis of herbal drugs and formulations. The results obtained from the study could be utilized for scientific validation and formulating standards for the quality assurance of the drug. The physicochemical properties of Juniperus virginiana [Table 1], [Table 2], [Table 3] help to identify and estimate the active compounds present in the drugs. In HPTLC fingerprinting, the developed chromatogram and Rf values of bands will be specific for the drug with the selected solvent system. UV spectroscopic study exhibits, four prominent peaks, which serve as characteristic standards.

Acknowledgment

The authors are thankful to Dr. Raj. K Manchanda, Director General, Central Council for Research in Homoeopathy, New Delhi for the encouragements facilities for the study: We acknowledge the assistance of Mr. T. S. S Reddy, Ex-SRF (Chemistry) during the study.

Financial Support and Sponsorship

Nil.

Conflict of Interest

There are no conflict of interest.[15]

 
  References Top

1.
Varma PN, Vaid I. Encyclopaedia of Homoeopathic Pharmacopoeia. Vol. II. New Delhi: B. Jain Publishers (P) Ltd.; 1997. p. 521.  Back to cited text no. 1
    
2.
Schwabe′s. A Compendium of Rare and Clinically Established Mother Tinctures. 3 rd ed. Noida: Dr. Willmar Schwabe India Pvt. Ltd.; 2010. p. 112.  Back to cited text no. 2
    
3.
Allen TF. The Encyclopedia of Pure Materia Medica. Reprinted Edition. New Delhi: B. Jain Publishers; 1982.  Back to cited text no. 3
    
4.
Anon. United States Homoeopathic Pharmacopoeia Convention. US Pharmacopeia (HPCUS). 1993.  Back to cited text no. 4
    
5.
Anon. Wealth of India, Raw Materials. New Delhi: Publications and Information Directorate, CSIR; 2010;5:310-11.  Back to cited text no. 5
    
6.
Hartwell JL, Johnson JM, Fitzgerald DB, Belkin M. Podophyllotoxin Juniperus species savinin. J Am Chem Soc 1953; 75:235-6.  Back to cited text no. 6
    
7.
Cushman KE, Maqbool M, Gerard PD, Bedir E, Lata H, Moraes RM. Variation of podophyllotoxin in leaves of eastern red cedar (Juniperus virginiana). Planta Med 2003;69:477-8.  Back to cited text no. 7
[PUBMED]    
8.
FAO. Flavours and Fragrances of Plant Origin. Food and Agriculture Organization of the United Nations; 2002.  Back to cited text no. 8
    
9.
Johansen DA. Plant Microtechnique. New York: McGraw Hill Book Co. Inc.; 1940.  Back to cited text no. 9
    
10.
Anon. Homoeopathic Pharmacopoeia of India. Vol. I. New Delhi: Ministry of Health and Family Welfare, Goverment of India; 1971.  Back to cited text no. 10
    
11.
Stahl E. Thin Layer Chromatography, A Laboratory Handbook. Berlin: Springer-Verlag; 1969.  Back to cited text no. 11
    
12.
Wagner H, Bladt S, Zgainski EM. Plant Drug Analysis, A Thin Layer Chromatography Atlas. Berlin: Springer-Verlag; 1984.  Back to cited text no. 12
    
13.
Sethi PD. High Performance Thin Layer Chromatography. New Delhi: CBS Publishers and Distributors; 1996.  Back to cited text no. 13
    
14.
Lacramiora I, Toma C, Rodica R. Histo-anatomical Researches Regarding Some Species of Cupressaceae. 2007. p. 34-7.  Back to cited text no. 14
    
15.
Anon., Standardisation of Single Drugs of Unani Medicine, Part I, New Delhi: CCRUM, Ministry of Health Family Welfare, Government of India. 1987.  Back to cited text no. 15
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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