|Year : 2013 | Volume
| Issue : 2 | Page : 41-46
Standardization of homoeopathic drug: Buxus sempervirens L.
P Subramanian1, P Padma Rao2, T Sheshashena Reddy1, P Sudhakar2, P Ramachandra Reddy3
1 Chemistry Section, Drug Standardisation Unit, O.U. B.32, Road No.4, Habsiguda, Hyderabad, India
2 Pharmacognosy Section, Drug Standardisation Unit, O.U. B.32, Road No.4, Habsiguda, Hyderabad, India
3 Department of Botany, Osmania University, Hyderabad, India
|Date of Submission||29-Jan-2013|
|Date of Acceptance||28-Jun-2013|
|Date of Web Publication||17-Aug-2013|
O.U.B. 32, Road No. 4, Habsiguda, Hyderabad - 500 007, Andhra Pradesh
Source of Support: None, Conflict of Interest: None
Background: Buxus sempervirens L. (Buxaceae), is a small tree, used in Homoeopathy for acute pain, increase in pulse rate and nausea. Leaves and stems are used in the preparation of medicine.
Objective : The pharmacognostic and physico-chemical studies are carried out to facilitate use of correct species and standard raw materials.
Material and Methods: Pharmacognostic studies of leaf and stem of authentic samples of Buxus sempervirens have been carried out. Physico-chemical parameters of the raw drug, namely, extractive values, ash value, formulation besides weight per milliliter, total solids, alcohol content, High Performance Thin Layer Chromatography (HPTLC) and Ultraviolet (UV) studies are given for the mother tincture.
Results: The leaves are nearly sessile, opposite, entire, narrowly lanceolate or ovate and up to 2.5 cm. The stomata are paracytic and confined to the abaxial side. Unicellular conical hair and peltate scaly hair occur on the adaxial side near the base. The midvein is ribbed on either sides. Crystalliferous idioblasts occur towards the abaxial side at the midvein and lamina. Secretory canals occur in the mesophyll. A single vascular bundle is present in the midvein. The stem is quadrangular. The vascular tissue is present as a cylinder with four cortical bundles, one each in the angles. The microscopical and organoleptic characteristics of the powder are provided.
Conclusion: The powder microscopic features and organoleptic characters along with anatomical and physico-chemical studies are diagnostic to establish the standards for ensuring quality and purity of the drug.
Keywords: Buxus sempervirens L., High performance thin layer chromatography, Homoeopathy, Pharmacognosy, Physico-chemical, Spectroscopy, Standardization
|How to cite this article:|
Subramanian P, Rao P P, Reddy T S, Sudhakar P, Reddy P R. Standardization of homoeopathic drug: Buxus sempervirens L. Indian J Res Homoeopathy 2013;7:41-6
|How to cite this URL:|
Subramanian P, Rao P P, Reddy T S, Sudhakar P, Reddy P R. Standardization of homoeopathic drug: Buxus sempervirens L. Indian J Res Homoeopathy [serial online] 2013 [cited 2015 Apr 25];7:41-6. Available from: http://www.ijrh.org/text.asp?2013/7/2/41/116618
| Introduction|| |
0Buxus sempervirens L., popularly known as "boxwood" in English; "Bachsbaum" in German; "Chikri" in Punjab and Kashmir; "Shamshad" in Urdu and Persian, is a small tree belonging to the family Buxaceae. It is a native of Western and Southern Europe and also occurs in Western Asia and North. Africa. In India, it is found in Himalayas and Punjab.  In Himalayas, it is found in Kumaon to Shimla, Punjab and Bhutan.  It is also cultivated as a hedge in gardens at Kodaikanal in Tamil Nadu.  Since it is grown at an altitude of 1600-2800 m, it is not found in other states.
The tincture of the young leaves and twigs are used as medicine in homoeopathy in the treatment for forcing pains (e.g., labor), frequent desire to urinate, increase in pulse rate and slight nausea.  In unani system of medicine, the leaves are used for the treatment of headache, pain and prolapse ani. The leaves are bitter, purgative, diaphoretic and useful in rheumatism and syphilis. 
In homoeopathy, its authority was mentioned in Br. Jour. Hom. 11, 158, for the effects of an infusion taken for the purpose of producing an abortion which it failed to do. 
Chemically the leaves are reported to contain buxenine-G (an alkaloid), sitosterol, stigmasterol, cycloartenol, lupeol, germanicol, β-amyrin, (+)-semperviramidine, (−) buxadienine (steroidal alkaloid), (+)-sempervirine, (−)-31-acteyl-cyclomicrophylline-A and (−)-benzoylbuxidienine from this plant. ,,,
A review of the literature reveals that no pharmacognostic standards have been recorded for the drug except for the anatomical review.  In view of the importance of the drug, pharmacognostic and physico-chemical studies of leaves and stem are carried out to lay down standards.
| Material and Methods|| |
The plant material of Buxus sempervirens L., was supplied by the Survey of Medicinal Plants and Collection Unit, The Nilgiris, Tamil Nadu. The leaves and young stems were fixed in formaldehyde-acetic acid-alcohol, dehydrated through the alcohol-xylene series, embedded in paraffin wax. Sections cut between 8 and 10 μm were stained in crystal violet - basic fuchsin combination.  The epidermal peels were obtained by gently scraping and peeling with a razor blade. The microscopic characters of powder were studied by boiling the powdered drug in distilled water, stained in saffranin and mounted in glycerine.
Preparation of Extracts
The air dried leaves and twigs of the drug were coarsely powdered to 10/44 (sieve size) and were subjected to the determination of moisture content (loss on drying at 105°C), total ash, water soluble ash, acid insoluble ash, extractability in different solvents, physico-chemical constants, thin layer chromatography (TLC) and ultraviolet aspects of mother tincture following official methods.  Mother tincture was prepared as per Homoeopathic Pharmacopoeia of India. 100 g of coarse powder of the drug was suspended in 680 mL of 95% alcohol and 350 mL of purified water for 24 hour at room temperature. It was filtered and made up to 1000 mL using same solvent ratio. Percolation method , was used for the preparation of mother incture.
High Performance Thin Layer Chromatography (HPTLC) Analysis
25 mL mother tincture was evaporated on water bath to remove alcohol. The residue was extracted with 3 mL × 25 mL chloroform. Concentrated chloroform extract was used for the HPTLC study. The concentrated chloroform extract was spotted in the form of the band of width 4 mm with a Camag microliter syringe on precoated silica gel aluminium 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 mL/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) at 254 nm and 366 nm by reflectance scanning and operated by Win Cats software (v 4.05, Camag) resident in the system. ,,
| Observations and Results|| |
A much branched shrub or small tree. Leaves are nearly sessile, opposite, narrowly lanceolate or ovate, up to 2.5 cm, entire, usually obtuse. Flowers small, yellow to green, strongly scented in small axillary heads or spikes. The terminal flowers are usually female, others being male. Capsule ovoid, 1.3 cm long, 3-horned, seeds 3-6, small. 
Leaves nearly sessile, opposite, narrowly lanceolate or ovate, up to 2.5 cm entire, usually obtuse, dark green above and pale beneath, glabrous. Stem quadrangular, 1.6-1.8 mm thick, covered by conical and occasionally few peltate scaly hair.
In surface view, epidermal cells are polygonal isodiametric in outline and have sides thin to slightly thick and straight to curved [Figure 1]a-c. The cells show scanty to slightly dense contents and are 5130/mm 2 (abaxial) and 7260/mm 2 (adaxial). The costal cells are anisodiametric to linear, parallelly oriented and are present on primary and secondary veins. The stomata are paracytic [Figure 1]c, and 260/mm 2 with stomatal index: 4.8; size 16-25 μm (19) long and 11-16 μm (14) wide.
|Figure 1: (a) Upper epidermis at the base with trichomes ×417. (b) Upper epidermis in surface (enlarged) ×364. (c) Lower epidermis in surface (enlarged) ×630 (Psh: Peltate scaly hair, uc: Unicellular conical hair, ps: Paracytic stomata)|
Click here to view
Trichomes are of two types: (1) Unicellular conical hair and (2) peltate scaly hair. The former are confined to the base, midvein and margins on adaxial [Figure 1]a and the latter are few and present more towards base, sides of midvein and near margins [Figure 1]a.
In a Transverse Section (TS), the midvein is ribbed on either sides and 216-346 μm (305) thick. The lamina is dorsiventral, 184-216 μm (200) thick [Figure 2]a. The margins are pointed and slightly bent inwards.
|Figure 2: (a)Transverse section (TS) of leaf at midvein ×162. (b) TS of leaf lamina ×132. (c)TS of stem ×145 (c: Cuticle, ue: Upper epidermis, le: Lower epidermis, p: Palisade tissue, vb: Vascular bundle, x: Xylem, ph: Phloem, cr: Crystal, sp: Spongy tissue, e: Epidermis, cb: Cortical bundle, pi: Pith, sc: Secretory canal)|
Click here to view
Epidermis is single-layered and composed of mostly barrel shaped cells, some isodiametric, oval to spherical. The epidermal cells are covered by a thick cuticle. Stomata are confined to lower surface of the leaf and flushed with epidermis. Unicellular trichomes are present on the adaxial surface and restricted to the base. Mesophyll is dorsiventral. Palisade is two-layered but at places three-layered, extending into midvein, cells 20-38 μm (30) long and 14-22 μm (17) wide, walls thin, contents dense with chloroplasts, occasionally with sphaeraphides. Spongy parenchyma is predominant with upper 3-4 layers of closely packed and lower portion loosely arranged with large intercellular spaces often interspersed with sphaeraphidal idioblasts. Some large secretory pores also occur in the mesophyll [Figure 2]a and b.
The ground tissue at midvein consists of 1-2 layered collenchyma on abaxial followed by 4-5 layers of parenchyma while the collenchyma on adaxial as a group of cells followed by 3-4 layers of parenchyma. Collenchyma cells are 8-16 μm (13) in diameter and angular. Parenchyma cells 8-25 μm (18) in diameter, often contain chloroplasts and interspersed with sphaeraphidal idioblasts mostly towards abaxial near vascular bundle, 14-36 μm (27) in diameter. Besides, few cells contain rhombic or hexagonal prismatic crystals of calcium oxalate [Figure 2]a.
The vascular bundle is single, large, oval, 227-281 μm (254) long and 162-205 μm (181) wide, endarch, conjoint, collateral and capped by lignified sclerenchyma. A layer of endodermis and pericycle encloses the vascular bundle. The xylem elements are numerous and arranged in radial rows. Phloem is extensive on the abaxial [Figure 2]a.
In TS, the stem is quadrangular with winged angles, covered by unicellular conical hair, papillate hair and peltate scales. The epidermis is single-layered covered by thick cuticle; hypodermis is collenchymatous as a group of cells, angular or lamellar in few with chloroplasts. Cortex is 8-10 layered in the interangles and 12-16 at the angles; cells polygonal to spherical and tangentially long; those at periphery collenchymatous, few with yellowish contents. Crystalliferous idioblasts occur in the phloem besides few in cortex. Cortical spherical vascular bundles are present one each in the angles [Figure 2]c.
Vascular tissue is in the form of thick secondary xylem cylinder. It is enclosed by an endodermis followed by pericycle. The phloem is precocious, external, encloses the xylem. A small amount of primary xylem is present close to pith. The xylem consisting of vessels or tracheids, fibres and xylem parenchyma are arranged in radial rows. In longisection, the vessels and tracheids show helical thickenings, besides few scalariform and bordered pits. Pith parenchyma is centrally present. Prismatic and rhombic crystals of calcium oxalate occur in the cortex close to cortical bundles while sphaeraphidal cells are close to the phloem [Figure 2]c.
- Pieces of adaxial epidermis.
- Pieces of abaxial epidermis with stomata.
- Unicellular hairs, broken, few.
- Rhombic and prismatic crystals of Calcium oxalate.
- Pieces of stem epidermis with parallelly oriented cells and attached unicellular hairs.
- Fragments of tracheary tissue with fibres.
- Fragments of leaf with epidermis and underlying palisade.
- Few sphaeraphidal crystals broken.
Colour: Moss green.
Odour: Slightly pungent.
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.
Results of physico-chemical studies are summarized in [Table 1], [Table 2] and [Table 3].
HPTLC Finger Printing
The profile of chromatographic separation scanned at 254 nm, reveals seven spots [Figure 3] and [Figure 4] out of which seven, six and four spots possess maximum composition with Rf at 0.86, 0.74 and 0.38 respectively. On the other hand scanned chromatogram at 366 nm revealed seven spots with seven, six and five spots showing maximum composition at Rf 0.89, 0.85 and 0.72 respectively. It is evident from the data that these are characteristic for the studied drug, which will help in the identification and authentication of the mother tincture. This is considered as valuable standards in pharmacopoeia. At 254 nm, seven spots appeared at Rf 0.15, 0.22, 0.29, 0.38, 0.52, 0.74 and 0.86 [Figure 3] and [Figure 4] with various concentrations while at 366 nm, seven spots at Rf 0.13, 0.27, 0.38, 0.51, 0.72, 0.85 and 0.89.
|Figure 3: High performance thin layer chromatography densitogram (chloroform: methanol [9:1 v/v]) of Buxus sempervirens mother tincture scanned at 254 nm|
Click here to view
|Figure 4: High performance thin layer chromatography finger printing (chloroform: methanol [9:1 v/v]) of Buxus sempervirens mother tincture scanned at 254 nm and 366 nm|
Click here to view
| Discussion|| |
The stomata have been reported to be confined to the lower surface and surrounded by rosettes of subsidiaries in Buxus. However, presently they are hypostomatic as has been reported earlier but presently they are distinctly paracytic in Buxus sempervirens studied. Further, the guard cells are strongly crested as also reported earlier.  The number of stomata are 260/mm 2 and the stomatal index is 4.8. The trichomes in Buxus were reported as unicellular conical  which is presently confirmed and are found restricted to the basal region near the midvein on the adaxial side. Besides, a few peltate scaly hairs are also found near the base on the adaxial side in the present species. Idioblasts containing prismatic crystals and sphaeraphides occur more on the abaxial side and also in the midvein as also reported earlier.  Some secretory canals are present in the mesophyll as also reported. 
In TS, the stem is quadrangular with winged angles. The epidermis is reported as thickly cutinized and papillate  which is presently confirmed. Besides, the epidermis possesses unicellular conical hair and rarely peltate scaly hair over its surface. The cortex is chlorenchymatous in its outer layer as also reported earlier.  The vascular tissue is a continuous vascular cylinder with four cortical bundles, one each in the angular wings. The presence of 1-2 layers of sclerenchyma fibres enclosing the cortical bundles has been described,  which is now confirmed. The xylem is a continuous cylinder exhibiting some secondary growth. The phloem is external and often possesses idioblasts with prismatic, rhombic and cluster crystals as also earlier reported.  The pith is quite abundant with thick walled pitted cells [Figure 2]c as also been reported earlier. 
Herbal medicines are composed of many constituents, therefore, amenable to variation. The physico-chemical analysis of Buxus sempervirens is presented in [Table 1], [Table 2] and [Table 3]. HPTLC fingerprinting is a precise and accurate method for herbal identification and can be used in authentication and characterization of this important medicinal plant. Hence, it is very important to obtain reliable chromatographic fingerprints that represent therapeutically active and chemically characteristic components of the herbal medicines. Thus, the developed chromatogram will be specific with selected solvent system and Rf value and serve as a good standardization tool for Buxus sempervirens.
Physical parameters include colour, appearance, odour, viscosity, moisture content, pH, sedimentation and ash values. Chemical parameters include limit tests, extractive values, chemical assays, etc. These standards along with the preparation of mother tincture furnished in Homoeopathic Pharmacopeia of India have been meticulously adhered to in our study.
| Conclusion|| |
The macro and microscopical, organoleptic characters along with the anatomical and methodology used for the studies are diagnostic and establish the standards. HPTLC analysis of Buxus sempervirens L. leaves and twigs can provide standard finger prints and will be used as reference tool for identification, authentication, quality control and standardization of this important medicinal plant.
| References|| |
|1.||Baburaj DS, editor. A Check List of Homoeopathic Medicinal Plants of India. Rev. ed. New Delhi: Central Council for Research in Homoeopathy; 2003. p. 43. |
|2.||Kirtikar KR, Basu D editors. Indian Medicinal Plants. Vol. 3. Dehradun: Bishen Singh Mahendrapal Singh; 1980. p. 2211-2. |
|3.||Hooker JD. The Flora of British India. London: L. Reeve and Co.;1890. p. 267. |
|4.||Allen TF. The Encyclopedia of Pure Meteria Medica. Reprint ed., Vol.2. New Delhi: B. Jain Publishers; 1982. p. 320. |
|5.||Atta-ur-Rahman, Dildar Ahmed, Asif S Erfan, Arshad Jamal, Iqbal Choudhary M, Bilge Sener, et al. Steroidal alkaloids from leaves of Buxus Sempervirens. Phytochemistry 1991;30(4):1295-8. |
|6.||Puckett RT, Sim GA. The structure of buxenine-G. Tetrahedron Lett 1966;32:3815-8. |
|7.||Atta-ur-Rahman, Dildar Ahmed, Bilge Sener, Songul Turkoz. Steroidal alkaloids from Buxus Sempervirens. Phytochemistry 1989; 28(4);1293-4. |
|8.||Abramson D, Goad JL, Goodwin TW. Triterpenes and sterols of Buxus sempervirens and local variations in their levels. Phytochemistry 1973;12:2211-6. |
|9.||Metcalfe CR, Chalk L. The Anatomy of the Dicotyledons. Vol. 2. Oxford: Clarendon Press; 1950. p. 1236-7. |
|10.||Johansen DA. Plant Microtechnique. New York: Macgraw Hill Book Co.; 1940. |
|11.||Homoeopathic Pharmacopeia Laboratory. Homoeopathic Pharmacopoeia of India. New Delhi: Controller of Publications; 1971. |
|12.||American Institute of Homoeopathy. Homoeopathic Pharmacopoeia of United States Convention: Chicago: Duncan Bros.;1993. |
|13.||Stahl E. Thin Layer Chromatography: A Laboratory Hand Book. Berlin: Springer-Verlag; 1969. |
|14.||Wagner H, Bladt S, Zgainski EM. Plant Drug Analysis: A Thin Layer Chromatography Atlas. Berlin: Springer-Verlag; 1996. |
|15.||Sethi PD. High Performance Thin Layer Chromatography. New Delhi: CBS Publishers and Distributers; 1996. |
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3]