Pharmacopoeial Compliance of Marketed Formulations containing seeds of Syzygium cumini (L.) Skeels

 

*Nartunai Govindarajan, Lokesh Ravichandran, Prem Kumar Chelladurai, Akansha Pandey, Vendal Murugesan, Ganji Kusuma

Captain Srinivasa Murthy, Central Ayurveda Research Institute, CCRAS, Ministry of AYUSH, Govt. of India,

A. A. Hospital Campus, Arumbakkam, Chennai – 600 106.

 

ABSTRACT:

Background: The seeds of Syzygium cumini are extensively used in Ayurveda, Siddha and Unani (ASU) systems of medicine to treat diabetes, polyuria, diarrhoea and vomiting. As per classical literatures and Ayurvedic Formularies, the seed kernel is to be used in the formulations. In practice, the food and herbal drug manufacturers are incorporating the whole seed along with testa instead of seed kernel, as such sold in raw drug market. This may leads to reduction in potency of drug in formulations. Objective: The study was carried out to confirm the Pharmacopoeial compliance for seeds of S. cumini in marketed formulations Methods: Macro-microscopic studies were carried out by following standard methods of official books. Results: Macroscopically the whole seed and seed kernel can be differentiated by colour, appearance, size and shape. Microscopically the seed kernel can be differentiated from whole seed by absence of sclereids and fibres. Conclusion: The findings will be helpful to analyse the Pharmacopoeial compliance of marketed formulations containing seeds of S.cumini and to differentiate the seed kernel from testa. The studied marketed formulations of S. cumini seeds in powdered form were found to be noncompliance with Pharmacopoeial standards, since whole seed powder along with testa was found instead of seed kernel as recommended.

 

 

 

INTRODUCTION: 

The plant Syzygium cumini (L.) Skeels Syn. Eugenia jambolana Lam. (Fam. Myrtaceae)¹ is commonly known as Jambu in Ayurveda²_, Jamun in Hindi, Naval in Tamil and Black plum or Jambolan in English³ (Fig. 1,2 &3). The seed kernel of Syzygium cuminiare extensively used in Ayurveda, Siddha and Unani (ASU) systems of medicine to treat diabetes⁴, polyuria⁴, intrinsic haemorrhage⁴, diarrhoea⁴ and vomiting⁴. As per classical literatures and AyurvedicFormularies, the seed kernel should be used in the formulations^4,5,6. The seed  kernel  of Syzygium cumini  is used in Ayurveda, Siddha and Unani formulations like Pusyanuga Curna⁶, Neerizhivu Choornam⁵, Aavaram vidhai Choornam⁵, Aavirai kudineer⁷, Safoof Ziyabetus⁸, Qurs Ziyabetus⁸ and Safoof Khasta⁸.

 

As single drug formulation, 3-6 g (unit dose) of powdered seed kernel is recommended in Ayurveda for the treatment of diabetes and polyuria². The formulations containing powdered seed are very popular in the market for its anti-diabetic activity.

 

 

Figure 1: Habit of Syzygium cumini (L.) Skeels

 

Figure 2: Inflorescence of Syzygium cumini (L.) Skeels	Figure 3:  Fruit twig of Syzygium cumini (L.) Skeels

 

The major phyto-constituents reported in seed kernel responsible for pharmacological activities are tannin³, gallic acid³ (Fig.4A), ellagic acid³ (Fig.4B), a glycoside (jamboline)³_, quercetin⁹, p-coumaric acid⁹, catechin⁹ (Fig. 4C), epicatechin⁹, caffeic acid⁹, tannic acid⁹ and pale-yellow essential oil³.

 

The important polyphenolic compounds such as gallic acid, ellagic acid and catechin are mainly responsible for major pharmacological activities. The seed kernel of S. cumini was reported to possess anti-diabetic, anti-bacterial, anti-oxidant, vibriocidal, anti-allergic, anti-nociceptive, anti-inflammatory and anti-fungal activities¹⁰ through pharmacological screening.

 

 

Figure 4: Chemical Structure of A: Gallic Acid, B: Ellagic Acid and C: Catechin

 

In this present study, the seeds were collected from field by taxonomic identification and marketed formulations of S. cumini seed powder were purchased from local market to establish its identity. The study was carried out to analyse the Pharmacopoeial compliance of seeds of S. cumini in powdered marketed formulations.

 

MATERIALS AND METHODS:

The seeds were collected from the Arignar Anna Hospital campus, Arumbakkam, Chennai (GPS Data-N13º4’40.449”, E80º13’5.654”) by taxonomic identification. The voucher specimen of the plant (No-0644A) and seeds (CSMCARI/F/SD-32) were deposited in Herbarium and Museum of CSMCARI, Chennai for future reference. The marketed single drug and compound formulations containing S. cumini seed powder were purchased from local market Chennai. The Macro-microscopic characterization was carried by following appropriate method published by standard/official books^{11,12,13,14,15}. The plant material were cleaned, properly dried, powdered and passed through sieve No.60 for powder microscopic study. The mounting and staining was carried out (phloroglucinol - 1%, iodine solution - 2% and safranin - 1%) by standard methods of prescribed books¹⁵. Plant transverse sections and powder characters were observed and reported as per guidelines and photographed under different magnifications with the help of Stereo zoom microscope (Zeiss Stemi 508) and Trinocular compound microscopes (Olympus BX43 and Zeiss Axiolab 5) fitted with Canon EOS 1500D camera and Axiocam 208 color camera. 

 

RESULTS:

The macroscopy, microscopy and powder characters of seed, seed kernel and marketed formulations were studied and the results are reported as follows.

 

Macroscopy:

The Macroscopical characters such as appearance, size & shape, colour, texture, odour and taste of the whole seed and seed kernel were studied (Figure. 5& 6). The results are reported in tabular form for easy comparison (Table.1).

 

 

Figure 5 : Dried Seeds and Seed kernels S. cumini

 

Figure 6. Dried Seed and Seed Kernel under stereo microscope of S. cumini

 

 

 

Table.1: Macroscopic characters of dried seeds and seed kernel of S. cumini

S. No	Macroscopic parameter	Dried whole seed	Dried Seed kernel
1.	Appearance	Seed kernel is enclosed by coriaceous covering, usually broader at the base and mucronate at the apex, Appears as fibrous surface under stereo microscope.	Rugged, cotyledons shows transversely and longitudinally connecting cracked lines, from where the cotyledon easily separable into 2-4 components
2.	Size & shape	Upto 2 cm long and 1cm in width, Ovoid to reniform	Upto 1.4 cm long and 0.8 cm in width Irregularly oval
3.	Colour	Cream with reddish brown patches	Black
4.	Texture	Rough	Rough
5.	Odour	Characteristic	Characteristic
6.	Taste	Astringent and slightly bitter	Astringent later slight bitterness

 

Microscopy:

Detailed TS of testa consists of wavyouter layer of epidermis, followed by 1 to 3 layers of sub-epidermis embedded with brownish content; followed by discontinuous ring of group of stone cells and sclereids; followed by a narrow, tangentially elongated obliterated parenchyma cells embedded with vascular strands; the innermost layer of the testa contains tangentially elongated, thin-walled, wavy parenchymatous cells with brownish content (Figure. 7).

 

 

Figure 7: Transverse section of Testa of S. cumini

Abbreviations: Brc-brown content cell, Epi-epidermis, Fib-fibres, Par-parenchyma Sc-sclereids and Vs-vascular strands .

 

Detailed TS of seed kernel shows a layer of outer epidermis with thin cuticle; followed by wide zone of mesophyll composed of polygonal, thin walled, parenchymatous cells fully packed with numerous starch grains, a few secretory cells at periphery, a few vascular strands scattered and cluster crystals of calcium oxalate (Figure. 8).

 

 

Figure 8. Transverse section of Seed Kernel of S. cumini

Abbreviations: Brc-brown content cell, Clr-cluster crystal of calcium oxalate, Epi-epidermis, Par-parenchyma, Sec-secretory cell, Sg-starch grains, Vs-vascular strands .

 

Powder microscopy:

The powder microscopic characters of testa and seed kernel (Figure.9) are studied separately and reported in Table 2.

 

Table 2: Powder Microscopic characters of testa and seed kernel of S. cumini

S. No.	Microscopic Characters	Testa	Seed Kernel
1.	Sclereids	Larger in size, Irregular, spindle shaped and elongated with mostly narrow lumen, walls striated prominently and pitted	Absent
2.	Fibres	Numerous, lignified, thick walled striated with blunt end and slightly pegged.	Absent
3.	Starch grains	Absent	Abundant starch grains, simple and compound, round to oval, central split like hilum, faint transverse striations, compound consists of 2 or 3 components, 5 to 45 µm in diameter
4.	Crystals	Absent	Cluster crystals of calcium oxalate ranging from 15 to 18 µm
5.	Vascular elements	Present	Present
6.	Brownish content	Present	Present

 

Figure 9: Powder Microscopic characters of Testa and seed kernel

Testa; A, Sclereid; B, Fibre; C, Vascular elements; D, Brownish content. Seed Kernel; a, Starch grains; b, Cluster crystals of calcium oxalate; c, Spiral vessel; d, Brownish content.

 

The transverse section of seed kernel shows presence of cluster crystals of calcium oxalate whereas the powder studied shows prismatic crystals. This is due to breaking of cluster crystal by impact force applied during powdering process.

 

As per the formulations of S. cumini is concerned, the Curna form are the most popular dosage form in the market. All the marketed single and compound formulations of S. cumini studied show the characters of whole seed instead of seed kernel (Figure.10).

 

DISCUSSION:

As per botanical nomenclature Eugenia jambolana Lam. Syzygium jambolanum (Lam.) DC is equated as accepted synonym for S. cumini¹⁶. As per recommendations of classical literature, the seed kernel which is devoid of testa is actually recommended in the Ayurvedic formulations.

 

The analytical standard for Syzygium cumini whole seeds has already been developed in API. In raw drug market the whole seeds are sold instead of recommended seed kernel as per classical literatures. In practice, the herbal drug and food manufacturers are incorporating unintentionally the whole seed along with testa in powdered formulations and this may lead to reduction in potency of drug in formulations. A study on comparative effectiveness of whole seed, seed kernel and testa reported that the seed kernel shows highest hypoglycaemic activity¹⁷. This may be due to presence high concentration of active ingredient tannin in seed kernel in comparison with whole seed and majority of the pharmacological screening reports were carried out by using the seed kernel¹⁸.

 

The ethyl acetate and methanol extract of seeds of S. cumini at the dose of 200-400mg/kg shows optimum anti diabetic activity by oral administration to experimental rabbits¹⁰. The probable mechanism of anti-diabetic activity of tannin present in seed kernel is by inhibiting alpha-glycosidase enzyme. It is reported that the antioxidant and anti-ulcerogenic properties of tannins present in seed kernel is responsible for gastro-protective and anti-ulcerogenic properties¹⁰.

 

The seed extracts of S. cumini was reported to have the following pharmacological activities such as anthelmintic activity¹⁹, inhibition of carbohydrates metabolizing enzymes²⁰, analgesic activity²¹, cytotoxic activity²², antibacterial²³, anti-diabetic activity^23,24 and antioxidant activity²⁵.

 

Figure 10: Powder microscopic key features of Marketed formulations 1, 2, 3, 4, 5 and 6

 

a, Powder; b, Sclereids; c, Fibres; d, Vascular elements; e, Starch grains; f, Cluster crystal of calcium oxalate; g, Brownish content.

Macro-microscopic characters of plant raw drugs play an important role in authentication and this should be the first and fundamental step to identify the botanical source. Even though monograph on seed kernel of S. cumini published in Ayurveda and Unani Pharmacopoeia of India, in this study the characters of testa and kernel are studied separately and photo-documented. The marketed single and compound formulations under study were found to contain the whole seeds instead of seed kernel only. The absence of sclereids and fibres in seed kernel can serve as a unique diagnostic character for single drug formulations. The presence of S. cumini testa in compound powdered formulations was confirmed by the characteristic nature of sclereids like larger size, spindle shape and prominently striated wall.

 

CONCLUSION:

The findings of this study will be helpful in authentication and differentiation of seed kernel and testa by macro-microscopical characteristics. Sclereids and fibres are found only in the testa and absent in seed kernel.  This diagnostic character plays a major role in differentiating whole seed and seed kernel in marketed formulations. The marketed single drug and compound formulations of S. cumini seeds in powdered form were analysed microscopically and found to be as whole seed powder along with testa instead of seed kernel. The results of this study throw light on Pharmacopeial noncompliance in use of S. cumini  seed in the powdered marketed formulations.

 

ACKNOWLEDGEMENT:

The authors are very grateful to the Director General, Central Council for Research in Ayurvedic Sciences, for his encouragement and providing opportunity to carry out the study.

 

CONFLICT OF INTEREST:

Nil.

 

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Accepted on 06.04.2023           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(12):5823-5827.