Micromorphological studies for application in the quality control of herbal medicines – some data of plants used in Mozambique

O. Silva1 and R. Serrano1 1Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa. Av. Professor Gama

Pinto, 1649-003 Lisbon, Portugal

Standardization and quality control are essential analytical tools to assure the correct identification of herbal raw materials in order to be used as herbal medicines. In this context, scientific work concerning the establishment of botanical biomarkers based on the major microscopic features observed using light microscopy and scanning electronic microscopy techniques on some of the most useful Mozambican medicinal plants were performed, including Hypoxis hemerocallidea Fisch. & C. A. Mey. (Hypoxidaceae) corm, locally known as “African potato” and Moringa oleifera Lam. (Moringaceae) leaf, a species native from north India actually extremely used at Mozambique and at others African countries as medicine and food and commonly known as “drumstick tree”, “horse radish tree” or “moringa”. Hereby, the principal results obtained by our team will be presented and allow the correct identification and usage of these two medicinal plants by the pharmaceutical and alimentary industries.

Keywords: Herbal medicines; Hypoxis hemerocallidea; micromorphological studies; Moringa oleifera; Mozambique; quality control.

1. Introduction

Standardization and quality control are essential analytical tools to assure the correct identification of herbal raw materials in order to be used as herbal medicines. Microscopy allows the identification of these herbal materials and the detection of individual components of a mixture [1]. For some well-known medicinal plants, data for the quality control were provided on official pharmacopoeias. However, for the most of plants used worldwide, such criteria are not found in these books. This fact is extremely important for example for the African continent, where knowledge of the use of medicinal plants remains essentially disclosed orally. During last sixteen years, our team has been developing scientific and pedagogical activities within the scope of the quality, preclinical safety and efficacy/mode of action of plants used in traditional medicine at Mozambique. These activities were carried out under institutional protocols with the Institute of Science and Technology of Mozambique (ISCTEM), the Centre for Research and Development in Ethnobotany (CIDE) and the Ministry of Health of Mozambique (MSM) [2]. Both Mozambican medicinal plants (Hypoxis hemerocallidea Fisch. & C. A. Mey. (Hypoxidaceae) corm, locally known as “African potato” and Moringa oleifera Lam. (Moringaceae) leaf, a species native from north India extremely used at Mozambique and on others African countries as medicine and food and commonly known as “drumstick tree”, “horse radish tree” or “moringa”, were used for treatment of different pathologies and have been object of different studies regarding their chemical composition, pharmacology and/or toxicology. However, although these two plants are set out in the African Herbal Pharmacopoeia [3], data related with the mandatory microscopy botanical identification within it, are scarce (African potato) or inexistent (moringa) and therefore it is relevant to deepen it. The aim of this chapter is to present and summarize the botanical biomarkers of these two herbal medicines, based on light microscopy (LM) and scanning electron microscopy (SEM) observations of samples obtained and studied in collaboration with the above mentioned Mozambican entities and already partially disclosed [4,5].

1.1 Hypoxis hemerocallidea Fisch. & C.A. Mey.

Hypoxis hemerocallidea Fisch. & C.A. Mey. (Hypoxidaceae) plants are geophytic and they overcome winter conditions in the form of an underground rootstock called “corm”. The plant grows well in warm and cold subtropical areas and the planting of the young corms should be done during the spring season. They also have adventitious roots attached to the corms that are thick and fleshy and which arise from the base of young corms. Aqueous infusions of this medicinal plant (African potato), are given to sickly children as a tonic, and to adults for dizziness and mental disorders, while fresh juice is applied to burn wounds [6]. It is traditionally used in Southern African countries to treat benign prostatic hyperplasia [7, 8] and more recently to treat AIDS [9]. A monograph of this medicinal plant is included in the African Herbal Pharmacopoeia as “Hypoxis hemerocallidea”, and the medicinal plant consists of the fresh or dried sliced corm of H. hemerocallidea Fisch. & C.A. Mey [10]. On this monograph the identification of the herbal drug is performed by means of botanical and thin layer chromatographic methods. However, illustrated morphological elements are scarce.

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1.2 Moringa oleifera Lam.

Moringa oleifera Lam. (Moringaceae), commonly known as “moringa” and as “behen tree, ben and benoil-tree, benzolive-tree, bred mouroung, brede mouroungue, drumstick tree, horseradish tree, mouroungue, tree of life” is native to sub-Himalayan regions of northern India and Pakistan but is now found throughout the tropics. Leaf, seed, bark, root, sap and flower of this species are used as food and/or medicine. A general description of the species, ethnobotanical information, chemical composition, pharmacological properties and the quality control methods by thin layer chromatography and NIR spectroscopy was described on the African Herbal Pharmacopoeia “Moringa oleifera” monograph [11] However, no data was described concerning the microscopic botanical identification of the different plant parts used as raw materials for pharmaceutical or alimentary uses. Although the bark, roots, leaves, and pods of this species are used for their medicinal properties, the most common is the leaf, that was reported to be a high value medicinal plant with different therapeutic uses [12], like anti-hypercholesterolemic [13] and antihypertensive [14] and anti-diabetes mellitus agent [15]. Reports have also described this part of the plant as a potent anti-inflammatory agent, with antitumor activity and hepatoprotective against anti-Mycobacterium tuberculosis drugs such as isoniazid and rifampicin [16]. Many other uses were described for the leaf of this species including: biogas production, green manure, domestic cleaning agent, bio-pesticide and as food [11, 17]. Results of in vivo studies involving aqueous leaf extracts, showed that this kind of preparations can be safe [12]. The leaf of the plant is commonly used on the dried form, whole or ground, or as dried extract based on dietary supplement formulations all around the world. It is therefore mandatory to establish clear quality rules for this plant material, including, among others, criteria for microscopic identification.

2. Material and Methods

2.1 Plant material

Corms of Hypoxis hemerocallidea Fisch. & C.A. Mey (Fig. 1 a) and leaves of Moringa oleifera Lam. (Fig. 1 b) were collected in Maputo (Mozambique) by the team of the Centre for Research and Development in Ethnobotany (CIDE). The voucher specimens are preserved in LISC Herbarium, Lisbon, Portugal. The plant material was dried in the dark under controlled conditions of humidity (75 ± 5%) and temperature (21 ± 1ºC) and preserved in the same conditions until use.

a) b)

Fig. 1 Macroscopic view of the plant material: a) Transverse section of H. hemerocallidea corm; b) Lower surface of M. oleifera leaf. Scale bars = 2000 µm a), and 5000 µm b)

2.2 Microscopic analysis

2.2.1 Light microscopy (LM)

The dried plant material was previously hydrated in water. For the anatomical analysis, each sample was sectioned freehand. Transverse sections and tangential longitudinal sections were cleared and mounted in a 60% chloral hydrate aqueous solution, according European Pharmacopoeia [18]. A representative portion of the total collected raw material selected to study was powdered using an Analytical Mill A-10 water-cooled laboratory mill (Staufen, Germany) and mounted in a 60% chloral hydrate aqueous solution, also according European Pharmacopoeia [18]. Microscopic analysis of the prepared corm and leaf sections and powdered plant material was conducted on an Olympus CX40 upright microscope (Tokyo, Japan), coupled with an Olympus ColorView IIIu camera (Tokyo, Japan). Image analysis was performed with Cell D 2006 Olympus Software (Tokyo, Japan).

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2.2.2 Scanning electron microscopy (SEM)

The dried plant material was sectioned, dehydrated at 35 ºC for 24 h and directly mounted on stubs using double-side adhesive tape. The samples were sputtered with a thin layer of gold in a JEOL JSM-1200 Fine Coater and observed in a JEOL JSM-T220 scanning electron microscope at 15 kV, with a digital image acquisition integrated system.

2.3 Quantitative data analysis

Stomatal index (SI) was determined by the following formula: SI , where (S) represents the number of stomata

in a given area of the leaf and (E), the number of epidermal cells in the same area of the leaf [18].

3. Results and Discussion

3.1 Hypoxis hemerocallidea

LM analysis of transversal sections of the H. hemerocallidea corm (Fig. 2) shows from the periphery to the interior, multiple layers of protective cork, also known as phellem, with brown content and formed from phellogen (cork cambium), followed by phelloderm cells tissue, and then parenchyma cells where was possible to find secretory ducts (Fig. 2 a). By SEM, in the cortical parenchyma it is observed the presence of sclereids with thick wall and narrow lumen (Fig. 2 b).

a) b)

Fig. 2 Transverse section of H. hemerocallidea corm: a) LM micrograph with parenchyma tissue containing secretory ducts (arrow); b) SEM micrograph of sclereids detail (arrow). Scale bars = 200 µm a), and = 50 µm b) Fig. 3 shows some characteristics structures, like calcium oxalate raphides crystals up to 80 μm long in parenchyma cells and many round starch grains with mostly 7-15 μm in diameter and occasional larger grains to 80 μm in diameter and a well-marked hilum.

a) b)

Fig. 3 SEM micrographs of H. hemerocallidea corm: a) Idioblast with calcium oxalate raphides crystals; b) Round starch grains in parenchyma cells. Scale bars = 10 µm a), and b) Microscopically, the powder of H. hemerocallidea corm is characterized by the presence of all the above-named features and by the presence of xylem reticulate vessels (Fig. 4).

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a) b)

Fig. 4 Xylem reticulate vessels in the powdered H. Hemerocallidea corm: a) SEM micrograph; b) LM micrograph. Scale bars = 50 µm a), and = 200 µm b)

3.2 Moringa oleifera

LM analysis of transversal sections of M. oleifera leaf (Fig. 5 a) shows an asymmetric organization with a mesophyll comprising two strata of palisade parenchyma and spongy parenchyma, with a small intercellular space volume on the lower epidermis, with a corresponding spongy parenchyma-palisade parenchyma ratio of 0.30. Uniseriate upper and lower epidermises are coated by a thick cuticle. The spongy parenchyma cells are irregular in shape and show occasionally crystalliferous idioblasts containing druses of calcium oxalate. Secretory ducts are present in the central part of the mesophyll, bellow palisade parenchyma cells (Fig. 5 b). Midrib transversal sections are typical with an amphicrival vascular bundle surrounded by collenchyma and xylem vessels radially arranged.

a) b)

Fig. 5 LM micrographs of M. oleifera leaf transverse section: a) Mesophyll asymmetric with palisade parenchyma, and spongy parenchyma with druses of calcium oxalate (arrow); b) Detail of a secretory duct. Scale bars = 100 µm a), and b) The leaf surface examination by LM (Fig. 6) and SEM (Fig.7) shows a slightly sinuous cuticle in both epidermises, a lower epidermis composed of polygonal to rectangular cells, a random stomata distribution (Fig. 6 a), and a upper epidermis with similar cells to those of the lower epidermis but they are usually smaller and the striations on the cuticle are sometimes less clearly marked (Fig. 6 b). Stomata anomocytic type (Fig. 6 and Fig. 7) are surrounded by a ring of six subsidiary cells and are more frequent in the lower surface. The calculated stomatal index (SI) is 4.7 on the upper epidermis and 12.6 on the lower epidermis.

a) b)

Fig. 6 LM micrographs of M. oleifera leaf surface view showing stomata anomocytic type: a) Lower epidermis; b) Upper epidermis. Scale bars = 50 µm a), and b)

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a) b)

Fig. 7 SEM micrographs of M. oleifera leaf surface view: a) Detail of a stomata with open ostiole and sunken below the lower epidermis; b) Anomocytic stomata with 6 subsidiary cells. Scale bars = 5 µm a), and = 10 µm b)

a) b)

Fig. 8 SEM micrographs of M. oleifera leaf surface view with unicellular covering trichomes, slightly curved: a) Short trichome; b) Long trichome. Scale bars = 10 µm a), and b) SEM analysis of M. oleifera leaf surface shows unicellular non-glandular trichomes (Fig. 8). Some are short and others long, but both are slightly curved and scarce on the lower epidermis. The powdered material of M. oleifera dried leaf is characterized by the presence of fragments containing the above-named structures.

4. Conclusions

This work emphasizes the importance of the use of micromorphological studies by LM and SEM for application in the quality control of herbal medicines. Obtained results are useful to allow the correct identification and usage of H. hemerocallidea corm and M. oleifera leaf as dried raw materials for industrial uses, including the alimentary and pharmaceutical ones. The set of characters observed and considered as typical for each of the medicinal plants under study on was summarized for the first time in the present work and can be the basis of a monographic text to be integrated in pharmaceutical quality monographs of each two important medicinal plants. Inclusion of the obtained results on the quality monographs of each medicinal plant is assumed to be extremely important, considering the increase in the marketing and consumption of products based on these two medicinal plants.

Acknowledgements The support by iMed.ULisboa (UID/DTP/04138/2013) from Fundação para a Ciência e a Tecnologia (FCT), Portugal, is gratefully acknowledged. A special thanks to all those who collaborated in the experimental work inherent to this publication, duly referred in References number [4] and [5] and also to the Centre for Research and Development in Ethnobotany (CIDE) of Mozambique.

References [1] Serrano R, da Silva G, Silva O. Application of Light and Scanning Electron Microscopy in the Identification of Herbal

Medicines. In: Microscopy: Science, Technology, Applications and Education, (Microscopy Book Series, Number 4), A. Méndez-Vilas & J. Díaz, editors, Vol. 1. Badajoz, Formatex Research Center; 2010. p.182-190.

[2] Silva O, Serrano R, Gomes ET. A Health an Educational Medicinal Plants Cooperation Project with Mozambique. In: Book of Abstracts 9th International Symposium on Chromatography of Natural Products, ISC NP. Lublin, Poland. May 26-29; 2014. p.178.

[3] Brendler T, Eloff JN, Gurib-Fakim A, Phillips LD. African Herbal Pharmacopoeia, Association for African Medicinal Plants Standards; 2010.

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[4] Taniça M, da Silva G, Lanzana MF, Agostinho A, Gomes ET, Serrano R, Silva O. Microscopic identification of Hypoxis hemerocallidea corm, an African traditional medicine. In Silva O, Serrano R, Chaves R, editors. Proceedings of the VIII International Ethnobotany Symposium, Lisbon, Portugal (October 3rd-8th; 2010); 2011. p. 497-512.

[5] Taniça M, da Silva G, Lanzana MF, Agostinho A, Gomes ET, Serrano R, Silva O. The application of micrographic parameters in the quality control of Moringa oleifera leaf. Planta Medica. 2010; 76:12.

[6] Buck AC. Phytotherapy for the prostate. BJU International. 1996; 78:325-336. [7] Pegel KH. Hipoxis Radix Plantzafrica. Extracta Urol. 1984; 1, suppl. 7:105-111. [8] Lowe FC, Ku J. Phytotherapy in treatment of benign prostatic hyperplasia: a critical review. Urology. 1996; 48:12-20. [9] Albrecht CF. Proceedings of the 2nd IOCD International Symposium, Victoria Falls, Zimbabwe. 25-28 February; 1996. [10] Association for African Medicinal Plants Standards (AAMPS), Hypoxis hemerocallidea, African Herbal Pharmacopoeia,

Brendler T, Eloff JN, Gurib-Farim A & Phillips LD editors, Mauritius, AAMPS; 2010. p.152-156. [11] Association for African Medicinal Plants Standards (AAMPS), Moringa oleifera, African Herbal Pharmacopoeia, Brendler T,

Eloff JN, Gurib-Farim A & Phillips LD editors, Mauritius, AAMPS; 2010. p.172-177. [12] Sidney JS, Hartman MJ, Review of the safety and efficacy of Moringa oleifera, Phytother. Res. 2015, 29, 796-804. [13] Ghasi S, et al. Hypocholesterolemic effects of crude extract of leaf of Moringa oleifera Lam in high-fat diet fed wistar rats. J. Ethnopharmacol. 2000; 69:21-25. [14] Faizi S, et al. Hypotensive constituents from the pods of Moringa oleifera. Planta Med. 1998; 64 (3):225-228. [15] Kar A, et al. Comparative evaluation of hypoglycaemic activity of some Indian medicinal plants in alloxan diabetic rats. J.

Ethnopharmacol. 2003; 84 (1):105-108. [16] Fakurazi S, et al. Moringa oleifera Lam. prevents acetaminophen induced liver injury through restoration of glutathione

level. Food Chem. Toxicol. 2008; 46:2611-2615. [17] Fahey JW. Moringa oleifera: a review of the medical evidence for its nutritional, therapeutic and prophylactic properties. Part 1.

Trees for Life Journal. 2005; 1:5. Available from www.tfljournal.org. Accessed 22/04/2016. [18] The European Directorate for the Quality of Medicines & HealthCare. In: European Pharmacopoeia 8th edition, EDQM

Council of Europe; 2013.

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