INTRODUCTION:

Quality control of herbal drugs has traditionally been based on appearance and today microscopic evaluation is indispensable in the initial identification of herbs, as well as in identifying small fragments of crude or powdered herbs, and detection of foreign matter and adulterants¹. A primary visual evaluation, which seldom needs more than a simple magnifying lens, can be used to ensure that the plant is of the required species, and that the right part of the plant is being used. At other times, microscopic analysis is needed to determine the correct species and/or that the correct part of the species is present.

For microscopic evaluation the dried parts of the plant are regarded as representative sample may require softening before preparation for microscopy by exposing the sample to a moist atmosphere either treated in watch glass, petri-plate or on a glass slide with appropriate organic solvent, or soaking in water. Bark, wood and other dense and hard materials usually need soaking in water for a few hours/overnight, until they are soft enough to be cut.

Boiling in water for a few minutes may also facilitate the cutting of the material. Any water-soluble contents may be removed from the tissues by soaking in water. Starch grains are gelatinized by heating in water.

Diabetes is especially more serious disease in India due to irrational food habits. But, most of the hypoglycemic agents and hypolipidemics used in allopathic practice to treat diabetes mellitus and hyperlipidemia are reported to have side effects in long term use². Hence, there is the need to search for effective and safe drugs for these ailments. Pharmaceutical research across the world shows that, natural products are potential sources of novel molecules for drug development³. A large number of polyherbal formulations are available for diabeties, which are used widely in all parts of the world.

Based on the above rationale the present study was undertaken with an aim to characterize some herbal antidiabetic drugs based on their microscopical characteristics and compare them with marketed formulations and in house developed formulations. The present paper reports on the characterization of herbal antidiabetic drugs based on microscopical characters like fragments of parenchyma cells, covering trichomes, fragments of vessels, Fragments of spirally thickened vessels, fragments of parenchyma cells filled with gelatinized starch, anomocytic stomata, paracytic stomata, fibrovascular bundles, rosette type calcium oxalate crystals, prism type calcium oxalate crystals, spiral and reticulate vessels, columnar palisade with externally thick cuticle cell flat at base, layer of covered thick walled cells containing alurone grains, oval rounded starch grains, tanniferous duct and some other special structures.

MATERIALS AND METHODS:

Plant material:

Following eight herbal anti diabetic drugs were chosen: Momordica charantia (seeds), Syzigium cumini (seeds), Trigonella foenum (seeds), Azadirachta indica (leaves), Emblica officinalis (fruits), Curcuma longa (rhizomes), Gymnema sylvestre (leaves) and Pterocarpus marsupium (heart-wood).

All eight herbs were procured from different parts of the state and were authenticated by Dr Anupam Pathak, Head, Department of Pharmacy, Barkatullah Vishwavidyalaya, Bhopal. A herbarium was submitted at the department. Herbarium number BUPH-4055.

Marketed samples:

Three market sample were selected and obtained from the local market-Baidyanath Madhumehari Churna (Batch no 217, Mfg-2/05, Baidyanath), Shivayu Madhuhari Churna (Batch no CH/4679, Mfg-1/05, Shiv Herbal Research Laboratories) and Meghdut Madhushoonya Churna (Batch no 104, Mfg-10/04, Meghdoot Gramodyog Sewa Sansthan). Characteristic microascopical structures were identified in individual crude drug powders, In-house preparations and marketed formulations.

Table 1: Ingredients for In-House Formulation-1 and as labeled in Baidyanath Madhumehari churna

Sr. No.	Ingredient	Quantity taken (Dry wt.)
1.	Gudmar	20 gm
2.	Jamun guthali	8 gm
3.	Karela beej	5 gm
4.	Haldi	5 gm
5.	Amla	5 gm
6.	Vijaysar	5 gm
7.	Tejpatra	5 gm
8.	Shilajeet	5 gm
9.	Kutki	4 gm
10.	Chitrak	4 gm
11.	Bilva patra	5 gm
12.	Trivanga bhasm	2 gm
13.	Methi	3 gm
14.	Neem patra	5 gm
15.	Other excipients (Pectin and Guar gum mixture)	q.s. to make 100gm

Preparation of In-House Formulation:

In house formulations were made in triplicate of the same proportions as in marketed products of each brand containing known amount of individual herbs. Formulation 1 was made in the same proportions as the marketed product of Baidyanath Madhumehari Churna containing known amount of individual herbs (Table 1). Formulation 2 was made in the same proportions as the marketed product of Shivayu Madhuhari Churna containing known amount of individual herbs (Table 2). Formulation 3 was made in the same proportions as the marketed product of Meghdut Madhushoonya Churna containing known amount of individual herbs (Table 3).

Table 2: Ingredients for In-House Formulation 2 and as labeled in Shivayu Madhuhari churna

Sr. No.	Ingredient	Quantity taken
1.	Gudmar	30 gm
2.	Karela Beej	10 gm
3.	Haldi	10 gm
4.	Jamun Guthali	10 gm
5.	Gudvel	5 gm
6.	Vijaysar wood	5 gm
7.	Babul Ki Chal	5 gm
8.	Maithi Beej	5 gm
9.	Bilva Patra	5 gm
10.	Amla	5 gm
11.	Neem Patra	5 gm
12.	Shilajeet	2.5 gm
13.	Trivanga bhasm	2.5 gm

Table 3: Ingredients for In-House Formulation 3 and as labeled in Meghdoot Madhushoonya churna

Sr. No.	Ingredient	Quantity taken
1.	Gudmar foil	15 gm
2.	Jamun seed	15 gm
3.	Karela seed	10 gm
4.	Amla	5 gm
5.	Maithi Beej	5 gm
6.	Asgandh	5 gm
7.	Haldi	5 gm
8.	Pure shilajeet	1 gm
9.	Neem Kauree	10 gm
10.	Gurhal pushp	5 gm
11.	Tulsi root	9 gm
12.	Vijaysar wood	15 gm

All the procured and authenticated individual drugs were dried in shade and cleaned by hand sorting. The individual drugs were then crushed using willing grinder and passed through mesh no. 40. The individual drugs were then weighed as per the quantity required. The drugs were mixed geometrically using double cone blender. The mixed formulation was unloaded, weighed and packed in labeled glass bottles.

Microscopic evaluation: ⁴

Slide preparation followed the classical method recommended by WHO (1998)^5-7. One or two drops of water, glycerol-ethanol or chloral hydrate were taken on a glass slide and the tip of a needle was moistened with water and a little quantity of the powdered material that adheres to the needle tip was taken. stir the powder in the drop of fluid was carefully stirred and applied on the cover-glass and slightly pressed to remove excess fluid from the margin of the cover-glass with a strip of filter-paper. The specimen was freed from air bubbles carefully by boiling the powder with the fluid on the slide until the particles were clear and the air was completely removed. After the slide was prepared micromorphology of the samples was studied by scintech trinocular research microscope in search of taxonomic characters. Anatomical measurements were made in ocular units at suitable magnification power and converted to digital photograph in high resolution 9X Olympus digital camera.

RESULTS AND DISCUSSION:

Haldi rhizome powder (C. longa):

Microscopy of curcuma showed abundant groups of parenchymatous cells, which were filled with gelatinised starch and permeated with a bright yellow colouring matter which was soluble in the aqueous mounts (Fig 1a). The covering trichomes were distinctively visible as unicellular, elongated, conical and bluntly pointed moderately thickened walls (Fig. 1b). The trichomes were found scattered and occasionally attached to fragments of the epidermis. The fragments of vessels in C.longa were abundant; they were mostly large and reticulately thickened with regularly arranged rectangular pits (Fig 1c). Some vessels having spiral or annular thickening were also visible.

Fig. 1 (A) Fragments of parenchyma cells filled with gelatinized starch in C. longa

Fig. 1(B) Covering Trichomes in C. longa

Fig. 1(C) Fragments of spirally thickened vessels in C.longa

Fig. 1 Different structures shown in the pure drug C.longa

Similar structures as mentioned above in the pure C. longa powder were also found in the prepared in house formulation (Fig. 2) and in the marketed polyherbal formulations of Madhushoonya (Fig. 3) and Madhuhari (Fig. 4).

Fig. 2 (A) Fragments of spirally thickened vessels in formulation

Fig. 2 (B) Fragments of parenchyma cells filled with gelatinized starch in formulation

Fig. 2 (C) Covering Trichomes in formulation

Fig. 2 Different structures shown in the In -House formulation

Neem leaves powder (A.indica):

The microscopy of powdered leaves of neem showed presence of thin and straight walled polygonal cells with anomocytic stomata The thick walled covering trichomes were found scattered in the epidermis. (Fig. 5 A and 5B). These structures were also seen in the in house formulation and marketed polyherbal formulations of Madhumehari (Fig. 6A and 6B), Madhuhari (Fig. 6C and 6D) and Madhushoonya (Fig. 6E and 6F.)

3 (A) Fragments of spirally thickened vessels in Madhushoonya

3 (B) Fragments of parenchyma cells filled with gelatinized starch in Madhushoonya

3(C) Covering Trichomes in Madhushoonya

Fig. 3 Different structures shown in the marketed formulation Madhushoonya

4 (A) Fragments of spirally thickened vessels in Madhuhari

4 (B) Fragments of parenchyma cells filled with gelatinized starch in Madhuhari

4 (C) Covering Trichomes in Madhuhari

Fig. 4 Different structures shown in the marketed formulation Madhuhari

Fig. 5 (A) Anomocytic stomata found in A. indica leaves powder

Fig. 5 (B) Thick walled covering trichome in A. indica leaves powder

Fig. 5 (C) Anomocytic stomata found in (Formulation)

Fig. 5 (D) Thick walled covering trichome (Formulation)

Fig. 6 (A) Anomocytic stomata found in Madhumehari (Baidyanath)

Fig. 6 (B) Fragment of covering trichome in Madhumehari (Baidyanath)

Fig. 6 (C) Anomocytic stomata found in Madhuhari (Shivayu)

Fig. 6 (D) Covering trichome in Madhuhari (Shivayu)

Fig. 6 (E) Anomocytic stomata found in Madhushoonya (Meghdoot)

Fig. 6 (F) Covering trichome in Madhushoonya (Meghdoot)

Microscopy of E. officinalis shows the presence of fibrovascular bundles structures, and rosette type calcium oxalate crystals (Fig. 7) prominently in the powder which confirms the sample. In the characterization of the prepared formulation and the marketed formulation similar structures were found in the different sample which confirms the presence of amla in prepared formulation and marketed formulations (Fig. 8 and 9).

Fig. 7 (A) Microscopy of E. officinalis – Fibrovascular bundles

Fig. 7 (B) Microscopy of E. officinalis- rosette type Calcium oxalate crystals

Fig. 7 Fragments of fiber - E. officinalis

Fig8 (A) Fibrovascular bundles (Formulation)

Fig. 8 (B) Fibrovascular bundles (Formulation)

Fig. 8(C) Rosette type Calcium oxalate crystals (Formulation)

Fig. 8 (D) Fibrovascular bundles (Formulation)

Karela fruit powder (M. charantia):

Microscopic study of momordica charantia mainly shows the presence of spiral and reticulate vessels and prism type calcium oxalate crystals as shown in Fig. 9 (A) and (B). During the characterization of prepared formulation and marketed formulation similar structures were shown under the microscope (Fig. 10 and 11).

Methi seeds powder (T. foenum):

Microscopic evaluation of T. foenum shows the presence of thick walled columnar palisade, covered externally with thick cuticle cell flat at base and pointed at apex and supported by tangentially elongated thin walled parenchymatus cells (Fig. 12). It also shows the presence of endosperm consists of layer of thick walled cells containing alurone grains and several layer of thin walled mucilaginous cell radially elongated and oil globules.

Fig. 9 (A)Rosette type Calcium oxalate crystal in Madhumehari (Baidyanath)

Fig. 9 (B) Fibrovascular bundles in Madhumehari (Baidyanath)

Fig. 9 (C) Rosette type Calcium oxalate crystals in Madhuhari (Shivayu)

Fig. 9 (D) Fibrovascular bundles in Madhuhari (Shivayu)

Fig. 9 (E) Rosette type Calcium oxalate crystals Madhushoonya (Meghdoot)

Fig. 9 (F) Fibrovascular bundles Madhushoonya (Meghdoot)

Fig. 10 (A) Spiral and reticulate vessels (M. charantia)

Fig. 10 (B) Prism type Calcium oxalate crystals (M. charantia)

Fig. 10 (C) Spiral and reticulate vessels in formulation

Fig. 10 (D) Prism type Calcium oxalate crystals in formulation

Fig. 11 (A) Spiral and reticulate vessels in Madhushoonya (meghdoot)

Fig. 11 (B) Prism type Calcium oxalate crystals in Madhushoonya (meghdoot)

Fig. 11 (C) Spiral and reticulate vessels in Madhuhari (Shivayu)

Fig. 11 (D) Prism type Calcium oxalate crystals Madhuhari (Shivayu)

Fig. 11 (E) Spiral and reticulate vessels Madhumehari (Baidyanath)

Fig. 11 (F) Prism type Calcium oxalate crystals Madhumehari (Baidyanath)

Fig. 12 (A) Endosperm consists of layer of covered thick walled cells containing alurone grains

Fig. 12 (B) Columnar palisade with externally thick cuticle cell flat at base

Fig. 12 (C) Endosperm consists of layer of thick walled cells containing alurone grains (formulation)

Fig. 12 (D) Columnar palisade covered externally with thick cuticle cell flat at base (formulation)

During the characterization of marketed formulations and in house prepared formulations, above mentioned structures were found which were similar to the microscopy of pure methi powder, indicating the presence of methi in all the formulation (Fig. 13). The microscopy of Madhuhari shows the above mentioned structures which are not very similar to the other structures as found in pure methi powder and different formulations. This may be due to extra bleaching.

Fig. 13 (A) Endosperm consists of layer of thick walled cells containing alurone grains Madhumehari (Baidyanath)

Fig. 13 (B) Columnar palisade covered externally with thick cuticle cell flat at base Madhumehari (Baidyanath)

Fig. 13 (C) Endosperm consists of layer of thick walled cells containing alurone grains (madhusoonya-Meghdoot)

Fig. 13 (D) Columnar palisade covered externally with thick cuticle cell flat at base (madhusoonya-Meghdoot)

Fig. 13 (E) Endosperm consists of layer of thick walled cells containing alurone grains (Madhuhari-Shivayu)

Fig. 14 (A) Oval rounded Starch grains found in the E. jambolana seed powder

Fig. 14 (B) Oval rounded Starch grains found in the prepared formulation powder

Jamun seed powder (E. Jambolana):

The microscopy of Eugenia shows the presence of brown coloured parenchymatus cells, numerous oval rounded starches (Fig. 14). Numerous oval rounded starches were seen in the prepared and marketed formulation (Fig. 15) confirming the presence of Eugenia in marketed products and prepared products. At the same time some specific structures were also found in the every powder that is seen in Fig. 16.

Fig. 15 (A) Oval rounded Starch grains found in the Madhumehari powder-Baidyanath

Fig. 15 (B) Oval rounded Starch grains found in the Madhusoonya powder-Meghdoot

Fig. 15 (C) Oval rounded Starch grains found in the Madhuhari powder-Shivayu

Fig. 16 : Special structures shown in (A) and (B) Pure Eugenia seed powder (C) Prepared formulation (D) Madhumehari (E) Madhuhari Powder (F) Madhusoonya powder

Vijaysar wood powder (P. marsupium):

Microscopy of vijaysar shows the presence of yellowish brown, plenty of lignified fibers, crystal fibers, reddish brown contents and free rhomboidal crystals of calcium oxalate crystals. Tannniferous ducts filled with red colour masses throughout phloem region. In the microscopic evaluation, rhomboidal crystals of calcium oxalate and tanniferous ducts were found in the powder of vijyasar as seen in Fig 17.

Fig. 17 (A) Rhomboidal crystals of calcium oxalate in P.marsupium

Fig. 17 (B) Tanniferous duct in formulation

Fig. 17 (C) Tanniferous duct in formulation

Fig. 17 (D) Rhomboidal crystals of calcium oxalate (formulation)

Similar structures to pure powder of vijaysar were also found in the microscopy of prepared in-house formulation and three marketed formulation that shows the presence of vijaysar in the prepared formulation and marketed formulation (Fig. 18, 19).

Fig. 18 (A) Tanniferous duct in Madhumehari-Baidynath

Fig. 18 (B) Rhomboidal crystals of calcium oxalate Madhumehari-Baidyanath

Fig. 18 (C) Tanniferous duct in Madhusoonya-Meghdoot

Fig. 18 (D) Rhomboidal crystals of calcium oxalate Madhusoonya-Meghdoot

Fig. 18 (E) Rhomboidal crystals of calcium oxalate Madhuhari-Shivayu

Fig. 18 (F) Tanniferous duct in Madhuhari-Shivayu

Fig. 19 Tanniferous duct and crystals -P.marsupium

Gudmar leaves powder (G. sylvestre):

Microscopy of gudmar shows the presence of light green epidermal cells, paracytic stomata, rosette crystals of calcium oxalate and broken pieces of trichomes and spiral vessels. In present study the microscopy of gudmar had shown the structures of broken pieces of trichomes and paracytic stomata in pure powder (Fig. 20). Similar structures of broken pieces of trichomes and paracytic stomata were also found in the prepared formulaion and marketed formulation (Fig. 21).

Fig. 20 (A)Paracytic stomata in G. sylvestre powder

Fig. 20 (B)Broken pieces of trichomes in G. sylvestre powder

Fig. 20 (C) Paracytic stomata in prepared formulation

Fig. 20 (D) Broken pieces of trichomes in prepared formulation

Fig. 21 (A)Paracytic stomata in Madhumehari -Baidyanath

Fig. 21 (B) Broken pieces of trichomes in Madhumehari -Baidyanath

Fig. 21(C) Broken pieces of trichomes in Madhushoonya-Meghdoot

Fig. 21(D) Paracytic stomata in Madhuhari-Shivayu

CONCLUSION:

The results obtained from the study could be utilized as a reference for microscopically examining the specific structures of the studied eight antidiabetic drugs individually and in a polyherbal formulation and can be used as a tool for reference standards for the quality control of anti diabetic drugs.

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Received on 21.06.2010 Modified on 03.07.2010

Research J. Pharm. and Tech. 4 (1): January 2011; Page 131-145