Phytochemical and anti-diabetic activities of different plant part extracts among black pepper (Piper nigrum) varieties in comparison with Piper longum and Piper betel

Table of contents

Table of figures

Table of tables

List of abbreviations

Phytochemical and anti-diabetic activities of different plant part among black pepper (Piper nigrum) varieties in comparison with Piper longum and Piper betel: an overview

Abstract

1. Introduction
1.1 Objectives
1.2 Scope of the study
1.3 Taxonomical classification: Piper nigrum
1.4 Taxonomical classification: Piper betle Linn.
1.5 Taxonomical classification: Piper longeum Linn

2. Review of literature
2.1 Uses and importance

3. Hypothesis

4. Materials and Methods
4.1 Study area
4.2 Sample collection and processing
4.3 Experiment design
4.4 Phytochemical screening
4.5 Determination of the changes in glucose level (Anthrone method)
4.6 Determination of the changes in glucose level (Benedicts test)
4.7 Statistical analysis

5. Results and discussion
5.1 Phytochemical screening
5.2 Glucose reduction tests

6. Conclusions

Acknowledgements

References

ACKNOWLEDGEMENTS

Firstly I thank God Almighty whose blessing were always with me and helped me to complete this project work successfully.

I am extremely grateful to my guide Dr. Prem Jose Vazhacharickal for his valuable guidance, co-operation, encouragement and motivation to carry out this project work successfully. I thank Dr. Sajeshkumar N.K and Jiby John Mathew for their valuable suggestions, support and encouragements. This research work will not be possible with the co-operation of many farmers. I express my sincere thanks to Mr. Binoy A Mulanthra (Lab in charge, Department of Biotechnology) for the support.

I wish to thank our beloved Manager Rev. Fr. Dr. George Njarakunnel, Respected Principal Dr. Joseph V.J, Vice Principal Fr. Joseph Allencheril, Bursar Shaji Augustine and the Management for providing all the necessary facilities in carrying out the study.

I am lovingly and gratefully indebted to my teachers, parents, siblings and my friends who were there always for helping me in this project.

Prem Jose Vazhacharickal*, Sajeshkumar N.K, Jiby John Mathew and Annie Babu

*Address for correspondence

Assistant Professor

Department of Biotechnology

Mar Augusthinose College

Ramapuram-686576

Kerala, India

Table of figures

Figure 1. Mean monthly rainfall (mm), maximum and minimum temperatures (°C) in Kerala, India (1871-2005; Krishnakumar et al., 2009)

Figure 2. Map of Kerala showing the various sample collection point of Piper nigrum, Piper longum and Piper betel. Authors own work

Figure 3. Pepper plant (Piper nigrum) immature peppercorns. Photo courtesy: Wikipedia

Figure 4. Black pepper (Piper nigrum) description a) tree bearing half mature fruits, b) plant climbing on support, c) spike and leaf, d) black, green, pink and white peppercorns, e) different types of peppercorns. Photo courtesy: Wikipedia

Figure 5. Black pepper (Piper nigrum) description a) black pepper grains, b) white pepper grains, c) peppercorn close-up, d) handheld pepper mills, e) roughly cracked black peppercorns. Photo courtesy: Wikipedia

Figure 6. Black pepper (Piper nigrum) description a) fully ripened fruits, b) mature fruits, c) green mature fruits separated from spike, d) red fully mature fruits separated from spike, e) mature and fully ripened fruits with spike, f) Dried peppercorns. Authors own images

Figure 7. Description of Narayakodi (P8) a) pepper plant with support tree b) pepper plant with spike and leaves, c) pepper plant leaf, d) spike with mature fruits, e) fully ripe fruits, spike without fruits, individual fruits. Authors own images

Figure 8. Description of Kotta (P10) a) and d) pepper plant with support tree, leaves and spike, b) mature spike with leaves, c) developing spike, e) mature spike with fruits. Authors own images

Figure 9. Description of Karimunda (P14) a) immature spike and fruits, leaves; b) and c) immature spike, d) and e) fully mature spike and fruit, f) mature spike. Authors own images

Figure 10. Description of Betal leaves (Piper betel; B) a) and e) leaves; b) cultivated plants on non living support; f) harvested Betal leaves on market. Photo courtesy: Wikipedia

Figure 11. Description of long pepper (Piper longum; T) a) fully mature spikes; b) semi mature spike on plant; c) and d) leaves; e) and f) developing spike. Authors own images

Figure 12. Piper nigrum L. lateral branch habit; 1-Errect, 2-Horrizontal, 3-Hanging (IPGRI, 1995)

Figure 13. Piper nigrum L. leaf lamina shape; 1-Ovate, 2-Ovate-elliptic, 3-Ovate-lanceolate, 4-Elliptic- lanceolate, 5-Cordate (IPGRI, 1995)

Figure 14. Piper nigrum L. leaf base shape; 1-Round, 2-Cordate, 3-Acute, 4-Oblique (IPGRI, 1995)

Figure 15. Piper nigrum L. leaf margin; 1-Even (entire), 2-Wavy (repand) (IPGRI, 1995)

Figure 16. Piper nigrum L. types of veining; 1-Acrodromous, 2-Campylodromous, 3-Eucamptodromous (IPGRI, 1995)

Figure 17. Piper nigrum L. spike orientation; 1-Errect, 2-Prostrate (IPGRI, 1995)

Figure 18. Piper nigrum L. spike shape; 1-Filiform, 2-Cylindrical, 3-Globular, 4-Conical (IPGRI, 1995)

Figure 19. Piper nigrum L. fruit shape; 1-Filiform, 2-Cylindrical, 3-Globular, 4-Conical (IPGRI, 1995)

Figure 20. Description of the powdered black peppercorns and plant parts of selected pepper varieties a) Narayakodi (P8) fruit, b) Narayakodi (P8) leaves, c) Narayakodi (P8) spike, d) Karimunda (P14) fruit, e) Karimunda (P14) leaves, f) Karimunda (P14) spike. Authors own images

Figure 21. Description of the powdered black peppercorns and plant parts of selected pepper varieties a) Kotta (P10) fruit, b) Kotta (P10) leaves, c) Kotta (P10) spike, d) betal leaves, e) Piper longum leaves, f) Piper longum spikes. Authors own images

Figure 22. Description of phytochemical analysis (Alkaloid; Anthocyanins; Anthroqinones; Carbohydrates; Coumarins; Emodins; Flavanoids; Glycosides; Lecoanthocyanins; Phenols; Phlobatannins; Proteins; Saponins; Tannins; Terpenoids; Control), of Karimuna variety (Fruit) in methanol extract. Authors own images

Figure 23. Description of phytochemical analysis (Alkaloid; Anthocyanins; Anthroqinones; Carbohydrates; Coumarins; Emodins; Flavanoids; Glycosides; Lecoanthocyanins; Phenols; Phlobatannins; Proteins; Saponins; Tannins; Terpenoids; Control), of Karimuna variety (Fruit) in petroleum ether extract. Authors own images

Figure 24. Description of phytochemical analysis (Alkaloid; Anthocyanins; Anthroqinones; Carbohydrates; Coumarins; Emodins; Flavanoids; Glycosides; Lecoanthocyanins; Phenols; Phlobatannins; Proteins; Saponins; Tannins; Terpenoids; Control), of Karimuna variety (Leaf) in methanol extract. Authors own images

Figure 25. Description of phytochemical analysis (Alkaloid; Anthocyanins; Anthroqinones; Carbohydrates; Coumarins; Emodins; Flavanoids; Glycosides; Lecoanthocyanins; Phenols; Phlobatannins; Proteins; Saponins; Tannins; Terpenoids; Control), of Karimuna variety (Leaf) in petroleum ether extract. Authors own images

Figure 26. Description of phytochemical analysis (Alkaloid; Anthocyanins; Anthroqinones; Carbohydrates; Coumarins; Emodins; Flavanoids; Glycosides; Lecoanthocyanins; Phenols; Phlobatannins; Proteins; Saponins; Tannins; Terpenoids; Control), of Karimuna variety (Spike) in methanol extract. Authors own images

Figure 27. Description of phytochemical analysis (Alkaloid; Anthocyanins; Anthroqinones; Carbohydrates; Coumarins; Emodins; Flavanoids; Glycosides; Lecoanthocyanins; Phenols; Phlobatannins; Proteins; Saponins; Tannins; Terpenoids; Control), of Karimuna variety (Spike) in petroleum ether extract. Authors own images

Figure 28. Description of glucose lowering activity using Benedict’s test of Narayakodi (fruit, leaf, spike) at 10 and 15% using methanol extract. Authors own images

Figure 29. Description of glucose lowering activity using Benedict’s test of Kotta (fruit, leaf, spike) at 10 and 15% using methanol extract. Authors own images

Figure 30. Description of glucose lowering activity using Benedict’s test of Karimunda (fruit, leaf, spike) at 10 and 15% using methanol extract. Authors own images

Figure 31. Description of glucose lowering activity using Benedict’s test of Betal leaves and long pepper (fruit, leaf) at 10 and 15% using methanol extract. Authors own images

Figure 32. Description of glucose lowering activity using Benedict’s test of Narayakodi (fruit, leaf, spike) at 10 and 15% using petroleum ether extract. Authors own images

Figure 33. Description of glucose lowering activity using Benedict’s test of Kotta (fruit, leaf, spike) at 10 and 15% using petroleum ether extract. Authors own images

Figure 34. Description of glucose lowering activity using Benedict’s test of Karimunda (fruit, leaf, spike) at 10 and 15% using petroleum ether extract. Authors own images

Figure 35. Description of glucose lowering activity using Benedict’s test of betal leaves and long pepper (fruit, leaf) at 10 and 15% using petroleum ether extract. Authors own images

Table of tables

Table 1. Different vernacular names of Piper nigrum L. around the globe and India

Table 2. Different vernacular names of Piper betel around the globe and India

Table 3. Different vernacular names of Piper longum around the globe and India

Table 4. Description of the Pepper nigrum L. varieties (P8, P14, P10) and places in Kerala

Table 5. Plant characteristics and properties of Piper nigrum L. varieties (P8, P14, P10) in Kerala

Table 6. Plant characteristics and properties of Piper nigrum L. varieties (P8, P14, P10) in Kerala

Table 7. Plant characteristics and properties of Piper nigrum L. varieties (P8, P14, P10) in Kerala

Table 8. Plant characteristics and properties of Piper nigrum L. varieties (P8, P14, P10) in Kerala

Table 9. Plant characteristics and properties of Piper nigrum L. varieties (P8, P14, P10) in Kerala

Table 10. Plant characteristics and properties of Piper nigrum L. varieties (P8, P14, P10) in Kerala

Table 11. Phytochemical analysis of Piper nigrum L. varieties, Piper betel, Piper longum and their plant parts in methanol extract

Table 12. Phytochemical analysis of Piper nigrum L. varieties, Piper betel, Piper longum and their plant parts in petroleum ether extract

Table 13. Antidiabetic activity study using different plant parts of Piper nigrum L. varieties (P8, P14, P10), Piper betel, Piper longum using anthrone method (methanol extract)

Table 14. Antidiabetic activity study using different plant parts of Piper nigrum L. varieties (P8, P14, P10), Piper betel, Piper longum using anthrone method (petroleum ether extract)

Table 15. Antidiabetic activity study using different plant parts of Piper nigrum L. varieties (P8, P14, P10), Piper betel, Piper longum using Benedict’s test (methanol extract)

Table 16. Antidiabetic activity study using different plant parts of Piper nigrum L. varieties (P8, P14, P10), Piper betel, Piper longum using Benedict’s test (petroleum ether extract).

List of abbreviations

illustration not visible in this excerpt

Phytochemical and anti-diabetic activities of different plant part among black pepper (Piper nigrum) varieties in comparison with Piper longum and Piper betel: an overview

Prem Jose Vazhacharickal1, Sajeshkumar N.K1, Jiby John Mathew1 and Annie Babu1

1Department of Biotechnology, Mar Augusthinose College, Ramapuram, Kerala, India-686576

Abstract

Diabetes mellitus is a clinical metabolic syndrome, which has been resulted from the malfunction or insufficiency of insulin production. Diabetes is the major threat to the people in developing and developed country. Prolonged diabetic condition can affect organs and synthetic drug if consumed by the patient can cause side-effects and impaired immune system. The synthetic medicine used for the treatment of diabetes is usually costlier, create side-effects and do not ensure complete recovery from the disorder but on the other hand, medicinal plants create a great impact in the human immune system. The medicinal plant produces a variety of chemical substances which shows significant therapeutic properties. This study highlights the potential anti-diabetic significance and phytochemical activity of different varieties Piper nigrum (Black pepper) and different strategies used for the assessment of these activities. The piperaceae family consist of 12 genera and more than 1400 species and is one of the largest families and widely distributed across the tropical and sub-tropical regions and over the high ranges of Andes and Sub Himalayas. Piper species naturally have multi-dimensional affect on various parts of the body often mentioned as antipyretic, diuretic, aphrodisiac, immune- stimulant and antioxidant, hepatoprotective, digestive, antiseptic and anti-diabetic. Phytochemical analysis of Methanol and Petroleum -ether extracts was carried out and different plant parts were collected from different districts across Kerala. Phytochemical which is identified from Piper nigrum present an exciting opportunity for the development of new type of therapeutics for diabetes mellitus. Some anti-diabetic plants exert their action by stimulating the function or number of beta cells and thus increasing insulin production with least side effects. Further investigations are required and more attention should be drawn to explore the biological activity of hundreds of traditionally used plants. Both in-vivo and in-vitro assessment is necessary for the assessment of anti-diabetic properties from the natural resources.

Keywords: Anti-oxidant; Piper nigrum; Phytochemical analysis; Non-insulin dependent diabetes mellitus.

1. Introduction

Black pepper (Piper nigrum) the flowering wine belongs to the family piperaceae, cultivated for its fruit, which is usually dried and used as spice and seasoning. Morphological characters like plant habit, pubescence, texture and leaf shape of juvenile and mature forms, orientation of the spike and length of peduncle, nature of bract and fruit colour are used as key distinguish different species ( Kanjilal et al.,1940; Gamble et al., 1925). Piperaceae is considered to be one of the most primitive families of Angiosperms (Engler, 1893; Rendle, 1925) derived from the herbaceous proto-angiosperms with simple, minute flowers (

Piperaceae is the large family consisting of herbs, shrubs and lianas (Sen et al., 2016; Jaramillo, 2001). The Piperaceae family consists of 12 genera and more than 1400 species (Trivedi et al., 2011) and is one of the largest families and widely distributed across the tropical and sub-tropical regions and over the high ranges of Andes and Sub Himalayas. In genus Piper more than 1000 are included, from which 110 are of the Indian origin (Parthasarathy, 2006; ). The largest germplasm conservatory is situated in IISR, Kozhikode (Ravindran, 2000). Piper betle, Piper longum and Piper cubeba are some the species which are economically important and used in indigenous medicine system and belong to the genus Piper of the family Piperaceae (Parthasarathy et al., 2006).

In India black pepper is cultivated to a large extent in Kerala, Karnataka and Tamil Nadu and to a limited extent in Maharashtra, North eastern states and Andaman and Nicobar Islands. Pepper cultivars grown in the provinces of Tellicherry and Malabar are known for their excellent aroma and pungency properties (Schulz et al., 2005). The white pepper of commerce is the product of the same pepper plant, produced by removing the pericarp (Ravindran, 2000). The various species of Piper leaves are aromatic and pungent to smell and plants possess secondary metabolites which are regarded as a part of its defence against plant-feeding insects and other herbivores (Parthasarathy et al., 2008). The small white flowers attached to the sessile are having pendulous, dense, slender spikes and the berries like fruits or peppercorns are normally of about 0.5-1.0 cm in diameter and contain a single seed while mature they become reddish-yellow or pale-yellow in colour but the young berries are green in colour, the length of the spike differs from varieties (Zachariah and Parthasarathy, 2008).The spices and herb naturally produce secondary metabolites which possess natural antioxidants that help in delaying of aging process and improve immune system (Nahak and Sahu, 2011).Piper species naturally have multi-dimensional affect on various parts of the body often mentioned as antipyretic, diuretic, aphrodisiac, immune stimulant, antioxidant, hepatoprotective, digestive, antiseptic and anti-diabetic (Trivedi et al., 2011). The wild forms of Piper nigrum are usually dioecious in nature and the bisexuals found are cultivated ones. They are native to tropical Asian regions mainly cultivated in India, Brazil, China, Africa, Sri-Lanka, Vietnam, Thailand, Indonesia and Malaysia for commercial and medicinal purposes (Ravindran, 2000). The roots of Piper nigrum has been used in Ayurvedic medicine and whole plant parts have been used for preparation of the medicated water for the bath (Mathew et al., 2003). Black pepper is often used to treat vertigo, asthma, chronic indigestion, colon toxins, obesity, sinusitis, congestion, fever, paralytic, arthritic disorders and also preferred to treat diarrhoea and cholera . It is often claimed that pepper consist antimicrobial activity (Dorman and Deans, 2000), antimutagenic (El-Hamss et al., 2003), anti-depressant (Lee et al., 2005), a free-radical scavenger (Gulcin,2005; Saxena et al., 2007), immuno-modulator, anti-tumor (Sunila and Kuttan, 2004), anti-thyroid (Panda and Kar, 2003), anti-diarrheal and anti-spasmodic (Bajad et al., 2001). The black pepper was reported to treat few common diseases like fever, cold, colic disorders and gastric conditions (Parmar et al., 1997; Kumar et al., 2007).

The Piper betel is an indigenous medicine plant and dioeciously in nature, perennial creeper which loves shade (Das et al., 2016). It has glossy heart shaped leaf with short adventitious rootlets, cultivated in the hotter and damper parts of the country and the size of the leaf varies with different varieties and cultivar from 7-15 cm in length and 5-14 cm in width, the leaves are alternate stipulate petiolate with 0.75-3.8 cm ovate cordate or obliquely elliptic, entire glabrous coriaceous 10-18 cm long and 5-10 cm broad acuminate oblique and rounded base 6-8 (Shukla et al., 2012). The betel is called in different names Pan (Hindi), Tambula (Sanskrit), Villayadela (Kannada), Vettila (Malayalam), Vettilai (Tamil), Tamala (Telugu), Nagarbel (Gujarati) (Das et al., 2016). The Piper betel has been categorized based on the morphological similarities and dissimilarities (). The leaves of the betel are propagated vegetative for commercial cultivation in large hectares (Das and Mallick, 2010). The different parts of Piper betel have been used such as leaves, roots, stems and stalks. The leaf is believed to possess anti-diabetic, anti ulcer, antiplatelet aggregation anti-tumour and respiratory depressant (. It is experimentally proved that the leaves possess diverse pharmacological actions (Nadkarni, 1982). It mainly has anti-inflammatory (Sarkar et al., 2008), anti-allergic activities (Wirotesangthong et al., 2008), hepatoprotective and anti-fertility (Manigauha et al., 2009).

The Piper longum L. (Piperaceae) is the slender aromatic climber and the whole plant is used for the treatment of carminative and analgesic (Lee et al., 2005). They are dioeciously in nature and both the male and female remain morphologically similar until the flowering stage (Manoj et al., 2005). The roots are perennial woody or remain as the creeper under the shrub and the branch lets are usually erect, glabrous with swollen nodes; the plant is attached to the tree with the roots clasping at the nodes; leaves are alternate, ovate, cordate, margin entire, glabrous and the mature female spikes are usually shorter and thicker when compared to the males of the same variety (Vikas et al., 2005; Oommen et al., 2000).The males are often found to be greenish-yellow, fleshy, cylindrical and having minute flowers, female spikes are erect and yellow (Manoj et al., 2004) .The spikes are edible and considered as frits are cylindrical, oblong, red or black when ripe, globule with aromatic odour and pungent to taste (Mohan et al., 2015). The Indian long pepper is know in different names such as Pipli, Pipar, Pipal (Hindi); Hippali, Thippali balli (Kannada); Tippali, Pippali (Malayalam); Pimpli (Marathi); Pippli,Tippli (Tamil); Pippallu, Pippali (Telegu); Magadhi, Kana, Ushana (Sanskrit) (Manoj et al., 2004; Oommen et al., 2000) . The fruits are void yellowish orange sunk in freshly spike and is frequently used in folk medicine to treat varieties of diseases like asthma, bronchitis, tumour disorders, jaundice, piles, insomnia, tuberculosis, leprosy, inflammations, spleen disorders the roots are used for abdominal pain, tumours, diseases of spleen (Jalalpure et al., 2003). The Piperine was the first amide isolated from the Piper species shows progressive effect in central nervous system depression, antipyretic and even anti-inflammatory activity hence used to treat mood disorders and moderate or mild depression states (Lee et al., 1984; Lee et al., 2005; Miyakado et al., 1979).

Black pepper requires humidity and high rainfall and it is largely cultivated in Kerala and favourable temperature range is 23-32°C (Zacharaiah et al., 2015). In India pepper is mainly cultivated in Karnataka, Kerala and Thamil Nadu (Seshachala et al., 2012). Pepper is mostly dioecious in wild form, but in the cultivated types the plants are mostly gynomonoecious or trimonoecious.(Thangaselvabal et al., 2012). A majority of the cultivated monoecious through variation in expression rainging from complete male to complete female is found. Over 75 cultivars of black pepper are being cultivated in India. Karimunda is the most popular cultivar in Kerala. The other importantcultivarsare Kottanadan, Narayakodi, Aimpiriyan, Neelamundi and Kuthiravally ( Devasahayam et al., 2013).

1.1 Objectives

The comparative study of different varieties of the pepper, betel leaves and long pepper was conducted to detect the changes in the activity of glucose level and the phytochemical constituents was determined using various chemical tests.

1.2 Scope of the study

The study highlights the potential anti-diabetic significance and phytochemical activity of different varieties of the Piper nigrum , Piper betel and Piper longum. Different strategies were used for the assessment of these activities. The traditional plant have astonishing capabilities to completely cure rare diseases like diabetes mellitus with least side-effects and does not impair the immune system of the patient when compared with the synthetic medicine popularly used. More studies in-vitro and in-vivo should be conducted for the assessment of the anti-diabetic properties of piperaceae families.

1.3 Taxonomical classification: Piper nigrum

Kingdom: Plantae-- planta, plantes, plants, vegetal

Subkingdom: Viridiplantae

Division: Tracheophyta – vascular plants, tracheophytes

Class: Magnoliopsida

Order: Piperales

Family: Piperaceae – peppers

Genus: Piper L.

Species: Piper nigrum L.

1.4 Taxonomical classification: Piper betle Linn.

Kingdom: Plantae-- planta, plantes, plants, vegetal

Subkingdom: Viridiplantae

Division: Tracheophyta – vascular plants, tracheophytes

Class: Magnoliopsida

Order: Piperales

Family: Piperaceae – peppers

Genus: Piper L.

Species: Piper betle Linn.

1.5 Taxonomical classification: Piper longeum Linn

Kingdom: Plantae-- planta, plantes, plants, vegetal

Subkingdom: Viridiplantae

Division: Tracheophyta – vascular plants, tracheophytes

Class: Magnoliopsida

Order: Piperales

Family: Piperaceae – peppers

Genus: Piper L.

Species: Piper longeum

Table 1. Different vernacular names of Piper nigrum L. around the globe and India.

illustration not visible in this excerpt

Table 2. Different vernacular names of Piper betel around the globe and India.

illustration not visible in this excerpt

Table 3. Different vernacular names of Piper longum around the globe and India.

illustration not visible in this excerpt

2. Review of literature

In black pepper high variability was also noticed for yield contributing characters like runner shoot production, holding capacity, adventitious root production, lateral branch habit, spike length, type of hermaphroditism, number of spikes per lateral branch, fruit set, dry weight, essential oil, oleoresin and piperine content. Intra-cultivar variability was reported earlier and was used to characterize the cultivars of black pepper (Ratnambal et al., 1985; Pillai et al., 1987; Ravindran et al., 1992; Ravindran et al., 1997; Sasikumar et al., 1999; Mathew and Mathew, 2002; Mathew et al., 2005).

Medicinal plants create a great impact in the human immune system. The primary healthcare of world’s 80% population are often met by the traditional medicines according to World Health Organization (WHO) (Duraipandiyan and Ignacimuthu 2006). The medicinal plant produces a variety of chemical substances which shows significant therapeutic properties (Ahmad and Beg, 2000; Iyengar, 1985; Chopra et al., 1992; Harborne and Baxter, 1995). The chemical substance carries out a definite physiological action on human body; the main active bioactive constituents of the plants are alkaloids, tannins, flavonoids and the phenolic compounds (Edeoga et al., 2005; Hill, 1952). The plants which consist therapeutic properties are often used as alternative treatments to health issues in developing countries (Duarte et al., 2005). Many indigenous medicinal plants are often used as the spices and food plants and referred to pregnant and nursing mothers for the medicinal purposes (Edeoga et al., 2005; Okwu, 1999; Okwu, 2001).

The spices are usually used as the supplementary herbs, besides hypoglycemic effects (Upasani et al., 2014). The diabetes mellitus is a metabolic disorder characterized by in appropriate hyperglycemia caused by a deficiency of insulin or resistance to its action at the cellular level (Dixit and Mittal, 2013; Alberti and Zimmet, 1998). Prolonged hyperglycemia can case disturbance in the metabolic process leading to serious complications like retinopathy, neuropathy, nephropathy, cardiovascular complications and even ulceration more than that it has indirect relations with many other diseases like endocrine disorders (Singab et al., 2014). The blood glucose level is controlled by both the insulin and glucagon, pancreatic endocrine hormones (Gupta and De, 2012).

The diabetes can be classified into many based on the etiology of the disease but widely accepted are two types type 1 and type 2 (Zimmet et al., 2004). The type 1; insulin dependent diabetes mellitus (IDDM) suffering persons have no capability or little capability to secrete insulin hence requires replacement therapy for the survival .The type 1 can be categorized into type 1a (90% of type 1) and type 1b (10% of type 1). Along with that few patients are susceptible to ketoacidosis with no evidence of developing autoimmune diseases (Betterle et al., 1984). The second category type 2; non insulin dependent diabetes mellitus (NIDDM), is characterized by the abnormality in the secretion of the insulin which gradually results in the weight loss with increase in the appetite and consumption of the food. These patients develop bladder, skin and vaginal infections with fatigue, nausea and vomiting (Gupta and De, 2012). The most common procedure of measuring the diabetes in blood is Fasting Plasma Glucose level (FPG), done in early morning; the Patients with FPG below 100 mg/dl are considered normal; 100-125 mg/dl indicates pre-diabetic condition and above 125 mg/dl are considered diabetic (Peters et al., 1996). The diabetes is chronic disorder which has become the third killer of the health of mankind along with cancer, cardiovascular and cerebrovascular (Li et al., 2004).

The deficiency in the production of insulin can be caused by the acquired or inherited deficiency hence the management of the diabetes is the global issue even after the development of synthetic medicine, the chances of the complete recover is comparatively less without side-effects (Malviya and Malviya, 2010; Li et al., 2004). Hence, most often alternative therapy involving the use of indigenous plants and herbal formulations are preferred by patients (Satyanarayana et al., 2006). The advantages of the herbs are easily available, can be consumed as raw, least side effects and low cost they are either a nutrient or a source of the food so it makes the king of all available therapies (Goel et al., 2012).

The black pepper is the herb which is often known as “Pippali” and used as antidiabetic polyherbal formulations (Upasani et al., 2014). The active alkaloid, piperine is present in Piper nigrum has been used for the evaluation of its efficacy to regulate the glucose level and its proven that oral administration for 15 days can lower the blood glucose level concentration and hepatic glucose-6-phospatase enzyme activity (Kohli et al., 2004).

2.1 Uses and importance

The Piper nigrum, Piper betel and Piper longum are widely and commonly used in the traditional medicines since the ancient times. The berries of the Piper nigrum are often used for the treatment of the intermittent fever, indigestion, cough, diarrhoea, asthma, diabetes, worm-infestation and it is often used as preservative agent due to its anti-oxidant and anti-microbial action (Chatterjee et al., 2007).

3. Hypothesis

The current research work is based on the following hypothesis

1) Various plant parts and solvent extracts of Piper nigrum L varieties shows difference in phytochemical activity.
2) The phytochemical activity of the Piper nigrum L have resemblance with other members of piperaceae.
3) The extracts of various parts of piperace family have different antidiabetic activity.

4. Materials and Methods

4.1 Study area

Kerala state covers an area of 38,863 km2 with a population density of 859 per km2 and spread across 14 districts. The climate is characterized by tropical wet and dry with average annual rainfall amounts to 2,817 ± 406 mm and mean annual temperature is 26.8°C (averages from 1871-2005; Krishnakumar et al ., 2009). Maximum rainfall occurs from June to September mainly due to South West Monsoon and temperatures are highest in May and November (Figure 1).

4.2 Sample collection and processing

Various pepper varieties in Kerala were selected based on a baseline survey, information’s collected from various beneficiaries and databases. Three different pepper varieties (Karimunda (Ka, P14), Kotta (K, P10), Narayakodi (N, P8)), betal leaves (Piper betel) and long pepper (Piper longum) were selected across Kerala for various phytochemical analysis and anti-diabetic activity. The pepper growing season is October and November and harvesting time is January and February. The fruit, leaves and spikes are collected from pepper plant and store in polyethylene zipper bags and processed in the laboratory. The samples were dried in hot air oven at 60ºC for 48hrs. The samples were finely powdered using a kitchen blender (Prestige Nakshatra plus, Prestige industries Mumbai) and later stored in air tight polyethylene zipper bag for analysis. The powdered samples were dissolved in two solvents: methanol and petroleum ether. The two concentration levels were selected includes 10 and 15% strength.

4.3 Experiment design

The experiment were conducted in a factorial manner, with three black pepper varieties (N, K and Ka); betal leaves (B); long pepper (T); three parts (Fruit; F, stem; S and leaf; L); two concentrations (10 and 15%); and two solvents (methanol and petroleum ether).

4.3.1 Experiment

The powdered samples were dissolved in the respective solvents with the two different concentrations. The dissolved extracts were transferred in polyethylene (PE) bottles.

4.4 Phytochemical screening

The chemical tests of phytochemical screening were carried out using the powered specimens and on aqueous extracts using the standardized procedure to analyze the constituents present in the samples as described by Banu and Cathrine (2015), Mathew et al. (2016) and Vazhacharickal et al. (2016).

4.4.1 Test for tannins

One ml of the plant extract was taken in a dry clean test-tube and one ml of the distilled water is gently poured with 0.5ml of five percentage ferric chloride .Observation of brownish green or a blue-black colouration indicate the presence of tannins.

4.4.2 Test for flavonoids

1.25 ml of dilute ammonia solution were added to a portion of the plant extract followed by the addition of concentrated H2SO4 .A yellow colouration observed in each extract indicated the presences of flavonoids. The yellow colouration disappeared on standing.

4.4.3 Test for terpenoids

1.25 ml of each extract was mixed with 0.5 ml of chloroform and concentrated H2SO4 (2-3 drops) was carefully added to form a layer. A reddish brown colouration of the interface was formed to show the positive results for the presence of terpenoids.

4.4.4 Test for saponins

2.5 ml of the extract was mixed with 1.25 ml distilled water was shaken vigorously for a stable persistent froth .The frothing was mixed with 1 drop of olive oil and shaken vigorously ,then observed for the formation of emulsion.

4.4.5 Test for phlobatannins

Deposition of red precipitate when the extract of each plant sample was boiled for an hour with one percentage aqueous hydrochloric acid was taken as evidence for the presence of phlobatannins

4.4.6 Test for carbohydrates

To a few ml of plant sample extract, two drops of Mayer’s reagent are added along the sides of test tube .Appearance of white creamy precipitate indicates the presence of alkaloids.

4.4.7 Test for glycosides

1 ml of plant extract was taken in a test tube and 1ml of chloroform and 1 ml acetic acid were added. Formation of violet to blue to green colouration shows the presence of glycosides

4.4.8 Test for coumarins

0.5 ml of the extract was taken in a test tube and 1.5 ml of 10% NaOH was added. Formation of yellow colour gives positive result to coumarins

4.4.9 Test for alkaloids

1 ml of plant extract was taken in a test tube and few drops of Mayer’s reagent were added. An appearance of white creamy precipitate shows positive result for alkaloids.

4.4.10 Test for phenols

The extract (5 mg) was dissolved in distilled water and 3ml of 10% lead acetate solution was added.

4.4.11 Test for emodins

Two ml of plant extract was taken in a test tube and two ml NH4OH and three ml of benzene were added .Formation of red colour indicates the presence of emodins.

4.4.12 Test for anthraquinones

Three ml of plant extract was taken in a test tube and three ml of benzene and five ml of ten percentage NH3 were added. Formation of pink, violet or red colouration in ammonical layer detect the presence of anthraquinones.

4.4.13 Test for anthocyanins

Two ml of plant extract was taken in a test tube and two ml of 2N HCl and NH3 were added. Formation of pinkish red to bluish violet colouration indicates the presence of anthocyanins.

4.4.14 Test for leucoanthocyanins

Five ml of isoamyl alcohol taken in a test tube and five ml of plant extract was added. Turn organic layer into red detects the presence of leucoanthocyanins.

4.5 Determination of the changes in glucose level (Anthrone method)

The amount of the soluble sugars is estimated using anthrone method using colorimetric techniques. The basic units of carbohydrates are monosaccharide which cannot be split by hydrolysis into simple sugars. Carbohydrates naturally exit as free sugars and polysaccharides. The anthrone reaction is rapid and convenient method for the determination of hexoses, aldopentoses and hexuronic acids either free or present in polysaccharides. Carbohydrates are dehydrated by conc.H2SO4 to form furfural. Furfural condenses with the anthrone to form blue-green coloured complex which can be measured easily colorimetrically at 630 nm.

4.6 Determination of the changes in glucose level (Benedicts test)

Benedicts test uses, Benedict reagent which is chemical reagent commonly used to detect the presence of reducing sugars. The reducing sugar is a carbohydrate which possesses either a free aldehyde or free ketone functional group as part of its molecular structure. The functional groups are the regions of a molecule that gives peculiar properties. A single molecule can have more than one functional group as the part of its structure. To conduct benedicts test 1ml of the sample is taken with the 3 ml of Benedict’s reagent and kept in boiling water bath for 5-10 minutes. As a result, glucose present in the sample gets reduces cupric (Cu++) ions to form a green to brick-red precipitate depending on the amount of sugar present.

4.7 Statistical analysis

The survey results were analyzed and descriptive statistics were done using SPSS 12.0 (SPSS Inc., an IBM Company, Chicago, USA) and graphs were generated using Sigma Plot 7 (Systat Software Inc., Chicago, USA).

illustration not visible in this excerpt

Figure 1. Mean monthly rainfall (mm), maximum and minimum temperatures (°C) in Kerala, India (1871-2005; Krishnakumar et al., 2009).

illustration not visible in this excerpt

Figure 2. Map of Kerala showing the various sample collection point of Piper nigrum, Piper longum and Piper betel. Authors own work.

illustration not visible in this excerpt

Figure 3. Pepper plant (Piper nigrum) immature peppercorns. Photo courtesy: Wikipedia.

illustration not visible in this excerpt

Figure 4. Black pepper (Piper nigrum) description a) tree bearing half mature fruits, b) plant climbing on support, c) spike and leaf, d) black, green, pink and white peppercorns, e) different types of peppercorns. Photo courtesy: Wikipedia.

illustration not visible in this excerpt

Figure 5. Black pepper (Piper nigrum) description a) black pepper grains, b) white pepper grains, c) peppercorn close-up, d) handheld pepper mills, e) roughly cracked black peppercorns. Photo courtesy: Wikipedia.

illustration not visible in this excerpt

Figure 6. Black pepper (Piper nigrum) description a) fully ripened fruits, b) mature fruits, c) green mature fruits separated from spike, d) red fully mature fruits separated from spike, e) mature and fully ripened fruits with spike, f) Dried peppercorns. Authors own images.

illustration not visible in this excerpt

Figure 7. Description of Narayakodi (P8) a) pepper plant with support tree b) pepper plant with spike and leaves, c) pepper plant leaf, d) spike with mature fruits, e) fully ripe fruits, spike without fruits, individual fruits. Authors own images.

illustration not visible in this excerpt

Figure 8. Description of Kotta (P10) a) and d) pepper plant with support tree, leaves and spike, b) mature spike with leaves, c) developing spike, e) mature spike with fruits. Authors own images.

illustration not visible in this excerpt

Figure 9. Description of Karimunda (P14) a) immature spike and fruits, leaves; b) and c) immature spike, d) and e) fully mature spike and fruit, f) mature spike. Authors own images.

illustration not visible in this excerpt

Figure 10. Description of Betal leaves (Piper betel; B) a) and e) leaves; b) cultivated plants on non living support; f) harvested Betal leaves on market. Photo courtesy: Wikipedia.

illustration not visible in this excerpt

Figure 11. Description of long pepper (Piper longum; T) a) fully mature spikes; b) semi mature spike on plant; c) and d) leaves; e) and f) developing spike. Authors own images.

illustration not visible in this excerpt

Figure 12. Piper nigrum L. lateral branch habit; 1-Errect, 2-Horrizontal, 3-Hanging (IPGRI, 1995).

illustration not visible in this excerpt

Figure 13. Piper nigrum L. leaf lamina shape; 1-Ovate, 2-Ovate-elliptic, 3-Ovate-lanceolate, 4-Elliptic- lanceolate, 5-Cordate (IPGRI, 1995).

illustration not visible in this excerpt

Figure 14. Piper nigrum L. leaf base shape; 1-Round, 2-Cordate, 3-Acute, 4-Oblique (IPGRI, 1995).

illustration not visible in this excerpt

Figure 15. Piper nigrum L. leaf margin; 1-Even (entire), 2-Wavy (repand) (IPGRI, 1995).

illustration not visible in this excerpt

Figure 16. Piper nigrum L. types of veining; 1-Acrodromous, 2-Campylodromous, 3-Eucamptodromous (IPGRI, 1995).

illustration not visible in this excerpt

Figure 17. Piper nigrum L. spike orientation; 1-Errect, 2-Prostrate (IPGRI, 1995).

illustration not visible in this excerpt

Figure 18. Piper nigrum L. spike shape; 1-Filiform, 2-Cylindrical, 3-Globular, 4-Conical (IPGRI, 1995).

illustration not visible in this excerpt

Figure 19. Piper nigrum L. fruit shape; 1-Filiform, 2-Cylindrical, 3-Globular, 4-Conical (IPGRI, 1995).

illustration not visible in this excerpt

Figure 20. Description of the powdered black peppercorns and plant parts of selected pepper varieties a) Narayakodi (P8) fruit, b) Narayakodi (P8) leaves, c) Narayakodi (P8) spike, d) Karimunda (P14) fruit, e) Karimunda (P14) leaves, f) Karimunda (P14) spike. Authors own images.

illustration not visible in this excerpt

Figure 21. Description of the powdered black peppercorns and plant parts of selected pepper varieties a) Kotta (P10) fruit, b) Kotta (P10) leaves, c) Kotta (P10) spike, d) betal leaves, e) Piper longum leaves, f) Piper longum spikes. Authors own images.

[...]