Piper longum (Linn.) restores ovarian function in Letrozole induced PCOS in Rats: Comparison with Metformin and Clomiphene citrate

Swati B. Pokale*, Dr. Ghanshayam Jadhav

Department of Pharmacology, Maratha Vidya Prasarak Samaj’s College of Pharmacy, MVP Campus, Gangapur Road, Shivaji Nagar, Nashik - 422002, Maharashtra, India.

*Corresponding Author E-mail: swati.18sept@gmail.com, aaryajadhav@gmail.com

ABSTRACT:

This study evaluates the prospective use of an herbal plant Piper longum in letrozole induced polycystic ovary syndrome using a rat model. The study used female wistar rats, which were divided into 9 groups, each containing 6 animals. Group I (Control) daily received 1% Carboxy Methyl Cellulose (CMC)as a vehicle control. Letrozole (1mg/kg) was administered by oral route for period of 21days for induction of PCOS in group (II-IX). During experimental period, vaginal smear of all females were collected daily for the estrous cycle determination. During 28 days of letrozole administration, changes in estrous cycle of females were observed and studied. This study showed that PCOS was induced. After Letrozole treatment, 6 animals from group III-IX treated orally with, standard drugs Metformin (300mg/kg/oral route), Clomiphene citrate (100mg/kg/oral route), plant drug Piper longum L. at a concentration of 200mg/kg, 400mg/kg and 800mg/kg, drug interaction groups: Metformin + Piper longum L, 800mg/kg and Clomiphene citrate +Piper lonum L., 800mg/kg body weight and studied for consecutive estrous cycles. Vaginal smear were examined, it showed that hydro alcoholic extract of fruits of Piper longum Linn. group has potential effect on PCOS bringing estrous cycle to normalcy. Also, after Letrozole treatment ovary and reproductive weights of normal rats increased which is normalizes with plant drug treatment. Further studies of hydroalcoholic extract of fruits Piper longum Linn. need to be carried out to check other related parameters of PCOS.

KEYWORDS: Polycystic ovarian syndrome, Hydro alcoholic extract of Piper longum Linn., Clomiphene citrate, Letrozole induced PCOS, Estrous cycle of rats.

INTRODUCTION:

Polycystic ovary syndrome (PCOS) is a one of current endocrinal disorder that affects women of childbearing age.¹ PCOS is a condition where women has imbalance of sex hormones leads to changes in menstrual cycle, formation of cyst and failure to conceive.² It is also known as Stein Leventhal Syndrome.^3-6 PCOS is usually a common health trouble which increases in adolescent girls along with young women for the period of the reproductive years.⁷ Planned teaching Programmes create awareness and improve knowledge for infertility and PCOS in adolescent girls.^8-10

The prevalence of PCOS varies with range of 2.2 and 26 %.^4,11 The common symptoms of PCOS include irregular menstrual cycles, weight gain and infertility.

The symptoms also include lack of ovulation, high androgen levels and high numbers of ovarian cysts. PCOS is a complex condition characterized by chronic absence of ovulation of polycystic ovary, clinical and biochemical hyperandrogenism, hirutism, amenorrhoea and infertility.¹² PCOS patient with obesity showed rise in insulin resistance and testosterone with normal body weight and ovulatory menstruation.^13,14 Treatment of PCOS is used for normalizing functions of ovary. Different medications are used for regulation of menstrual cycles and to stimulate ovulation as they contain different pharmacological active components with promising effects in PCOS. As per WHO, approximately 80% of world’s population uses different herbal medicines due to accessibility and affordability.¹⁵ now, a day’s various drugs in addition to aerobic exercise are used for treatment of PCOS still effective treatment to control PCOS is challenge. Use of different herbal plant products is the only source to prevent and cure various diseases.^16,17

Piper longum Linn., Piperaceae, is growing in warmer regions of India, Western ghats, Central Himalaya to Asam, Bengal known as Pipali in Sanskrit, Long pepper in English, and Thippilli in Tamil. Fruits are small, avoid, sunken in fleshy spike, 2.5-4cm long, blackish green and shinning. Volatile oil, saponins, carbohydrates, starch, proteins, alkaloids, and amygdalin are present in fruits but no tannins. The alkaloids isolated from roots and stems were identified as piperine, piperlongumine and methyl-3,4,5-trimethoxy-cinnamate. In Indian traditional medicines, Piper longum L. has been used for centuries for treatment of various inflammatory, diabetic, ulcer disorders.^18-21

MATERIALS AND METHODS:

Collection of plant material:

Fruits of Piper longum Linn were collected during Sep- Oct from Pirangut area in Pune, Maharshtra, India was authenticated by Dr. C. R. Jadhav, Botanist, Botanical Survery of India, Ministry of Environment, Forests and Climate Change, Koregoan Road, Pune and a voucher specimen (BSI/WRC/IDEN.CER./2029/H3) was preserved in our laboratory, Maratha Vidya Prasarak Samaj’s College of Pharmacy, Nashik.

Preparation of Hydro alcoholic extract:

The fruits of Piper longum (Linn) were collected and shade dried completely under normal conditions to maintain its active principles and secondary metabolites present in it. Dried fruits were reduced to coarse powder using mixer grinder. These powdered fruits were stored in tight container. Powdered fruits of Piper longum Linn. were extracted separately with 1000ml of diluted alcohol (70:30-Alcohol: Water) by heating under reflux on water bath for 6 hours at 55°C. The mixture was then filtered and marc was extracted twice again in same manner. The filtrates from each extracted step were pooled and concentrated under vacuum using a rotary vacuum evaporator. After complete extraction, the extract was stored sterile bottles under refrigeration conditions, until further use.²²

Animals:

Female Wistar rats weighing (200-250gms) were obtained from Crystal Biological Solutions, Handewadi, Pune. They were maintained at controlled room temperature (20°C - 22°C) and relative humidity (30%- 70%) with 12:12 hr light and dark cycle. All the animals were fed with commercially available normal pellet diet (NPD) and water ad libitum during the course of an experiment. All the experimental procedures and protocols used in study were reviewed and approved by Institutional Animal Ethics Committee (Protocol No: MVPCP/IAEC/2019/05) of Maratha Vidya Prasarak Samaj’s College of Pharmacy, Nashik constituted under Committee for Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Environment and Forests, Government of India. Ethical guidelines were strictly followed during all the experimental period.

Experimental Groups and Study Design:

Female Wister rats of 150-200gms were randomly divided into 9 groups (n=6 /group), as first control group that received vehicle only (1% carboxymethyl cellulose) once daily p.o and other treated. Before staring experiment, all female wistar rats were acclimatized for 14 days to laboratory conditions. The eight treatment groups rats were administered with Letrozole (LETROZ TAB Manufactured by Sun Pharma India, Ltd) at concentrations of 1.0mg/kg p.o. dissolved in 1% CMC (2.0mg/kg) once daily.^23-24 The second disease control group, PCOS induced was left for natural recovery for 15 days post Letrozole treatment. The third and fourth PCOS induced groups were treated repetitive dose of 300mg/kg daily p.o. Metformin (GLYCOMET Manufactured by USV Pvt. Ltd., Kumrek, Sikkim, India) and 100mg/kg daily p.o. Clomiphene citrate (SIPHENE- 100 Manufactured by Serum Institute of India Pvt. Ltd., India) which are known modern drug used in the treatment of polycystic ovary syndrome.^25,26 The fifth, sixth and seventh PCOS induced groups were treated with repetitive doses of 200mg/kg, 400mg/kg and 800 mg/kg of hydro alcoholic extract of fruits of Piper longum L. once daily p.o. for 35 days. Eight group, PCOS induced group was treated with Piper longum L. 800mg/kg daily p.o. and Metformin 300mg/kg daily p.o. while ninth group was treated with Piper longum L. 800 mg/kg daily p.o. and Clomiphene citrate 100mg/kg/ orally/daily for 35days. During, treatment period, a vaginal smear was obtained to monitor phases of estrous cycle of each group. On 15^th day after plant drug treatment all rats were sacrificed for further evaluation.¹⁶

Examination of estrous cycle:

A clean fresh cotton bud was dipped in normal saline. It is then inserted softly in the vaginal notch of the female rats as well as a fresh swab was obtained. This cotton bud was rolled on a clean grease free slide for making a smear and kept to air dry. Added few drops of methanol and fixed the cells in the smear. The slide was air dried. Giemsa stain was added to smear and covered the slide. The slide was set aside covered in the petridish for 5 minutes. After 5 minutes distilled water was added to the giemsa and gently rocked. On the top of the slide a green scum was appeared. The slide was stained for 10 minutes in dilute giemsa. The stained slide was drained to remove excess stain and then washed in gentle stream of tap water. The washed slide was air dried and observed under the microscope in 40x objective.¹⁶

RESULTS:

The various estrous cycle patterns were examined using vaginal smear method to detect PCOS in rats (Table No.1). Normal estrous cycle of rats lasts for 4-5 days which consists of four basic phases in sequential order: estrous, metaestorus, diestrous and proestrous (Figure No. 1). Ovarian weights as well as reproductive system weights of all nine groups were compared (Table No. 2). PCOS induction period showed that during estrous cycle examination, Letrozole treated rats had irregularity in estrous cycle phases (Table No. 3 and Figure No. 2). Estrous cycle irregularity changed to normal sequence after treating rats with plant drug Piper longum Linn and also compared with Metformin and Clomiphene citrate (Table No. 4 and Figure No. 3).

Figure 1: Estrous cycle of Rat

Table No. 1: Representing protocol of Study design

Treatment	Group I Control	Group II Disease control Letrozole	Group III Letrozole + Metformin	Group IV Letrozole + Clomiphene citrate	Group V Letrozole + Piper Longum 200mg/kg
1%CMC	1-28 days	---	---	---	---
Letrozole	1-28 days	1-28 days	1-28 days	1-28 days	1-28 days
Metformin	---	---	29-63 days	---	---
Clomiphene citrate	---	---	---	29-63 days	---
Plant drug, Piper longum L.					29-63 days
Piper longum + Metformin	---	---	---	---	---
Piper longum +Clomiphene citrate	---	---	---	---	---
Natural recovery	---	29-63 days	---	---	---

Table No. 1: Continue

Treatment	Group VI Letrozole + Piper Longum 400mg/kg	Group VII Letrozole + Piper Longum 800mg/kg	Group VIII Letrozole + Piper Longum 800mg/kg + Metformin	Group IX Letrozole + Piper Longum 800mg/kg + Clomiphene citrate
1%CMC	---	---	---	---
Letrozole	1-28 days	1-28 days	1-28 days	1-28 days
Metformin	---	---	---	---
Clomiphene citrate	---	---	---	---
Plant drug, Piper longum L.	29-63 days	29-63 days	29-63 days	29-63 days
Piper longum + Metformin	---	---	29-63 days	---
Piper longum +Clomiphene citrate	---	---	---	29-63 days
Natural recovery	---	---	---	---

Table No. 2: Representing weight of rats reproductive system

Parameters

Group I Control

Group II Disease control Letrozole

Group III Letrozole

+ Metformin

Group IV Letrozole + Clomiphene citrate

Group V Letrozole +

Piper Longum 200mg/kg

Ovary weight/body (gm)

0.49gm

0.50gm

0.45gm

0.46gm

0.44gm

Weight of female reproductive system (gm)

2.233gm

3.243 gm

2.160 gm

2.172 gm

1.971 gm

Table No. 2: Continue

Parameters

Group VI Letrozole

+ Piper Longum 400mg/kg

Group VII Letrozole

+ Piper Longum 800mg/kg

Group VIII Letrozole + Piper Longum 800mg/kg

+ Metformin

Group IX Letrozole + Piper Longum 800mg/kg

+ Clomiphene citrate

Ovary weight /body (gm)

0.43gm

0.44gm

0.45gm

0.46gm

Weight of female reproductive system (gm)

1.859 gm

1.912 gm

2.081 gm

2.074 gm

Table No. 3: Comparison of estrous cycle of all groups during PCOS induction

Days	Group I	Group II	Group III	Group IV	Group V
1	Estrous	Metaestrous	Estrous	Diestrous	Metaestrous
2	Estrous	Metaestrous	Metaestrous	Diestrous	Metaestrous
3	Estrous	Metaestrous	Metaestrous	Diestrous	Metaestrous
4	Metaestrous	Diestrous	Metaestrous	Diestrous	Diestrous
5	Metaestrous	Diestrous	Metaestrous	Proestrous	Diestrous
6	Metaestrous	Diestrous	Metaestrous	Proestrous	Proestrous
7	Diestrous	Diestrous	Diestrous	Proestrous	Proestrous
8	Diestrous	Proestrous	Diestrous	Estrous	Proestrous
9	Proestrous	Proestrous	Proestrous	Estrous	Metaestrous
10	Proestrous	Proestrous	Proestrous	Metaestrous	Metaestrous
11	Proestrous	Proestrous	Proestrous	Metaestrous	Metaestrous
12	Estrous	Metaestrous	Metaestrous	Metaestrous	Metaestrous
13	Estrous	Metaestrous	Estrous	Diestrous	Metaestrous
14	Metaestrous	Metaestrous	Metaestrous	Diestrous	Diestrous
15	Metaestrous	Metaestrous	Metaestrous	Proestrous	Diestrous
16	Metaestrous	Metaestrous	Metaestrous	Proestrous	Diestrous
17	Diestrous	Diestrous	Diestrous	Proestrous	Proestrous
18	Diestrous	Diestrous	Diestrous	Estrous	Proestrous
19	Proestrous	Proestrous	Proestrous	Metaestrous	Proestrous
20	Proestrous	Proestrous	Proestrous	Metaestrous	Proestrous
21	Proestrous	Proestrous	Proestrous	Metaestrous	Proestrous
22	Estrous	Metaestrous	Proestrous	Metaestrous	Metaestrous
23	Estrous	Metaestrous	Proestrous	Diestrous	Metaestrous
24	Estrous	Metaestrous	Proestrous	Diestrous	Diestrous
25	Metaestrous	Metaestrous	Metaestrous	Diestrous	Diestrous
26	Metaestrous	Metaestrous	Metaestrous	Proestrous	Proestrous
27	Diestrous	Diestrous	Diestrous	Proestrous	Proestrous
28	Diestrous	Diestrous	Diestrous	Proestrous	Proestrous

Table No. 3: Continue

Days	Group VI	Group VII	Group VIII	Group IX
1	Estrous	Estrous	Metaestrous	Diestrous
2	Metaestrous	Metaestrous	Metaestrous	Diestrous
3	Metaestrous	Metaestrous	Metaestrous	Proestrous
4	Metaestrous	Metaestrous	Diestrous	Proestrous
5	Metaestrous	Diestrous	Diestrous	Proestrous
6	Metaestrous	Diestrous	Proestrous	Proestrous
7	Diestrous	Proestrous	Proestrous	Proestrous
8	Diestrous	Proestrous	Proestrous	Proestrous
9	Proestrous	Proestrous	Metaestrous	Estrous
10	Proestrous	Estrous	Metaestrous	Estrous
11	Proestrous	Estrous	Metaestrous	Metaestrous
12	Estrous	Metaestrous	Metaestrous	Metaestrous
13	Estrous	Metaestrous	Diestrous	Metaestrous
14	Metaestrous	Metaestrous	Diestrous	Metaestrous
15	Metaestrous	Diestrous	Diestrous	Diestrous
16	Metaestrous	Diestrous	Proestrous	Diestrous
17	Diestrous	Proestrous	Proestrous	Proestrous
18	Diestrous	Proestrous	Proestrous	Proestrous
19	Proestrous	Proestrous	Proestrous	Proestrous
20	Proestrous	Proestrous	Estrous	Proestrous
21	Proestrous	Estrous	Metaestrous	Metaestrous
22	Proestrous	Metaestrous	Metaestrous	Metaestrous
23	Metaestrous	Metaestrous	Diestrous	Metaestrous
24	Metaestrous	Metaestrous	Diestrous	Metaestrous
25	Metaestrous	Diestrous	Proestrous	Diestrous
26	Metaestrous	Diestrous	Proestrous	Diestrous
27	Diestrous	Proestrous	Metaestrous	Proestrous
28	Diestrous	Proestrous	Metaestrous	Proestrous

Table No. 4: Comparison of estrous cycle of all groups during Drug treatment

Days	GroupI	Group II	GroupIII	GroupIV	Group V
29	Proestrous	Proestrous	Proestrous	Proestrous	Proestrous
30	Proestrous	Proestrous	Proestrous	Metaestrous	Metaestrous
31	Proestrous	Proestrous	Proestrous	Metaestrous	Metaestrous
32	Estrous	Metaestrous	Proestrous	Metaestrous	Metaestrous
33	Estrous	Metaestrous	Metaestrous	Diestrous	Metaestrous
34	Metaestrous	Metaestrous	Metaestrous	Diestrous	Metaestrous
35	Metaestrous	Metaestrous	Metaestrous	Proestrous	Diestrous
36	Metaestrous	Metaestrous	Metaestrous	Proestrous	Diestrous
37	Diestrous	Diestrous	Diestrous	Proestrous	Proestrous
38	Diestrous	Diestrous	Diestrous	Metaestrous	Proestrous
39	Proestrous	Proestrous	Proestrous	Metaestrous	Proestrous
40	Proestrous	Proestrous	Proestrous	Metaestrous	Metaestrous
41	Proestrous	Proestrous	Proestrous	Metaestrous	Metaestrous
42	Estrous	Metaestrous	Metaestrous	Metaestrous	Metaestrous
43	Estrous	Metaestrous	Metaestrous	Diestrous	Metaestrous
44	Metaestrous	Metaestrous	Metaestrous	Diestrous	Diestrous
45	Metaestrous	Metaestrous	Metaestrous	Proestrous	Diestrous
46	Metaestrous	Metaestrous	Metaestrous	Proestrous	Proestrous
47	Diestrous	Diestrous	Diestrous	Proestrous	Proestrous
48	Diestrous	Diestrous	Diestrous	Estrous	Proestrous
49	Proestrous	Proestrous	Proestrous	Estrous	Estrous
50	Proestrous	Proestrous	Proestrous	Estrous	Estrous
51	Proestrous	Proestrous	Proestrous	Metaestrous	Estrous
52	Estrous	Metaestrous	Estrous	Metaestrous	Metaestrous
53	Estrous	Metaestrous	Estrous	Diestrous	Metaestrous
54	Estrous	Metaestrous	Estrous	Diestrous	Diestrous
55	Metaestrous	Metaestrous	Metaestrous	Proestrous	Diestrous
56	Metaestrous	Metaestrous	Diestrous	Proestrous	Proestrous
57	Diestrous	Diestrous	Diestrous	Estrous	Proestrous
58	Diestrous	Diestrous	Proestrous	Estrous	Estrous
59	Proestrous	Proestrous	Proestrous	Estrous	Estrous
60	Proestrous	Proestrous	Proestrous	Metaestrous	Estrous
61	Estrous	Metaestrous	Estrous	Metaestrous	Metaestrous
62	Estrous	Metaestrous	Estrous	Metaestrous	Metaestrous
63	Estrous	Metaestrous	Estrous	Diestrous	Metaestrous

T No. 4: Continue

Days	Group VI	Group VII	Group VIII	Group IX
29	Proestrous	Proestrous	Metaestrous	Proestrous
30	Proestrous	Proestrous	Metaestrous	Metaestrous
31	Proestrous	Metaestrous	Metaestrous	Metaestrous
32	Proestrous	Metaestrous	Diestrous	Metaestrous
33	Metaestrous	Metaestrous	Diestrous	Metaestrous
34	Metaestrous	Metaestrous	Proestrous	Metaestrous
35	Metaestrous	Diestrous	Proestrous	Diestrous
36	Metaestrous	Diestrous	Proestrous	Diestrous
37	Diestrous	Proestrous	Estrous	Proestrous
38	Diestrous	Proestrous	Estrous	Proestrous
39	Proestrous	Proestrous	Metaestrous	Proestrous
40	Proestrous	Estrous	Metaestrous	Proestrous
41	Proestrous	Estrous	Metaestrous	Proestrous
42	Estrous	Metaestrous	Diestrous	Metaestrous
43	Metaestrous	Metaestrous	Diestrous	Metaestrous
44	Metaestrous	Metaestrous	Proestrous	Metaestrous
45	Metaestrous	Diestrous	Proestrous	Diestrous
46	Metaestrous	Diestrous	Estrous	Diestrous
47	Diestrous	Proestrous	Estrous	Proestrous
48	Diestrous	Proestrous	Estrous	Proestrous
49	Proestrous	Proestrous	Estrous	Estrous
50	Proestrous	Estrous	Metaestrous	Estrous
51	Proestrous	Estrous	Metaestrous	Estrous
52	Estrous	Estrous	Metaestrous	Metaestrous
53	Estrous	Metaestrous	Diestrous	Metaestrous
54	Metaestrous	Metaestrous	Diestrous	Metaestrous
55	Metaestrous	Diestrous	Proestrous	Metaestrous
56	Metaestrous	Diestrous	Proestrous	Diestrous
57	Diestrous	Proestrous	Estrous	Diestrous
58	Diestrous	Proestrous	Estrous	Proestrous
59	Proestrous	Estrous	Estrous	Proestrous
60	Proestrous	Estrous	Estrous	Estrous
61	Estrous	Estrous	Metaestrous	Estrous
62	Estrous	Metaestrous	Metaestrous	Estrous
63	Estrous	Metaestrous	Metaestrous	Metaestrous

Figure 2: Comparison of estrous cycle of groups during PCOS induction

Figure 3: Comparison of estrous cycle of groups during Drug treatment

DISCUSSION:

During study period, various estrous cycle patterns were observed in Control (Group I) and other treated groups (Group II to IX). Group I showed all four patterns for 63 days while Group II showed with three different patterns as metaestrous, diestrous and proestrous; lack of estrous phase due to PCOS condition. Persistent vaginal cornification (PVC) was preceded for irregular estrous cycles.²⁷ As per references, the relative proportion of epithelial, cornified cells and leukocytes to evaluate the estrous cycle.²⁸ Group III showed all four patterns for PCOS induction period for 28 days along with absence of estrous phase with treatment Metformin up to 35 days. Group IV showed all four patterns for 63 days with increased proestrous phase indicating irregular estrous cyclicity reflected. Group V plant hydro alcoholic extract of fruits of Piper longum Linn. group 200mg/kg showed three different patterns as metaestrous, diestrous and proestrous; lack of estrous phase for 28 days but all four patterns for 63 days with repetitive occurrence of metaestrous and estrous phases. Group VI plant hydro alcoholic extract of fruits of Piper longum Linn. group 400mg/kg showed presence of all four patterns for 63 days with increased metaestrous phase representing irregular estrous cyclicity reflected. It was identified that approximately 4-5 days of regular estrous cycle in study animal rats consists of estrous, metestrous, diestrous and proestrous phases and the average pregnancy period lasted for 21 days (19-23 days).²⁹ Group VII of plant hydro alcoholic extract of fruits of Piper longum Linn. group 800mg/kg showed irregular phases to normal sequence of estrous cycle. Group VIII plant hydro alcoholic extract of fruits of Piper longum Linn. group 800mg/kg in combination with Metformin and Group IX plant hydro alcoholic extract of fruits of Piper longum Linn. group 800mg/kg in combination with Clomiphene citrate showed all the patterns of estrous phases.

CONCLUSION:

Normal estrous cycle lasts for 4-5 days in rats. First phase is estrous (9-15 hrs) showed presence of cornified epithelial cells (Figure 1a), second phase is metaestrous (10-14 hrs) showed leucocytes with cornified cells (Figure 1b), third phase is diestrous (60-70 hrs) showed only presence of leucocytes (Figure 1c) and fourth phase is proestrous (12 hrs) which is a preparatory step for next estrous cycle (Figure 1d). In Polycystic ovary syndrome, irregular estrous cycles were observed in rats and preceded by persistent vaginal cornification method. Animals treated with hydro alcoholic extract of fruits of Piper longum Linn. group restores estrous cycle to 80% normalcy compared with combination groups. Thus, hydroalcoholic extract of fruits of Piper longum Linn. group has potential effect on PCOS bringing estrous cycle to normalcy. Also, after Letrozole treatment ovary and reproductive weights of normal rats increased which is normalizes with plant drug treatment. Further studies of hydroalcoholic extract of fruits Piper longum Linn. Need to be carried out to check other related parameters of PCOS.

ACKNOWLEDGMENT:

Authors are grateful to Maratha Vidya Prasarak Samaj’s College of Pharmacy, MVP Campus, Gangapur Road, Shivaji Nagar, Nashik and Crystal Biological Solutions, Pune for providing all the facilities to complete this research work.

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Received on 31.12.2020 Modified on 07.02.2021

Research J. Pharm. and Tech. 2021; 14(10):5190-5196.

DOI: 10.52711/0974-360X.2021.00903