Elucidation of the Probable Ovarian-Dependent Mechanism of the Estrogenic Effects of Buccholzia coriacea and Progesterone Effects of Cogniauxia pololeana in the Rat ()
1. Introduction
In Congo Brazzaville, traditional medicine uses plants to cure different kinds of diseases and prevent pregnancy; without however having scientific data on their pharmacological efficacy and mechanism of action. According to [1], two plants in the traditional Congolese pharmacopoeia, namely Buchholzia coriaceaEngl (Capparidaceae) and Cogniauxia podolaena Baill (Cucurbitaceae) are frequently used by the population for their curative virtues.
In addition, the literature reports that the decoction of Cogniauxia podolaena leaves is used in Brazzaville to treat infertility [2]. Also [3] [4] reported that the plant drug from the leaves of Cogniauxia podolaena is forbidden to pregnant women because of its abortifacient power. As for Buchholzia coriacea, it is reportedly used in traditional Nigerian medicine for the treatment of amenorrhoea [5].
Pharmacological studies carried out on the aqueous extracts of Cogniauxia podolaena have highlighted their anti-diabetic [6], anti-plasmodial and cytotoxic [7], and analgesic [8] activities. Buchholzia coriaceaextracts have also been studied in the laboratory for anti-inflammatory, analgesic and antipyretic [9], antibacterial and antifungal [10], hypoglycaemic and antioxidant activities [11].
According to [12] [13] Buchholzia coriaceaand Cogniauxia podolaena block the sexual cycle of the rat at the estrus and di-estrus stages respectively, thus demonstrating their oestrogenic and progesterone effects in mammals. It is known that sex hormones are essentially produced by the ovaries; administration of these hormones in the absence of the ovaries makes it possible to correct certain ovarian deficiencies in castrated animals. It is in this context that the present study was conducted, which consists of evaluating the effects of aqueous extracts of the leaves of Buchholzia coriaceaand Cogniauxia podolaena in castrated rats in order to elucidate the probable ovary-dependent mechanism of action of these two plants.
2. Material and Methods
2.1. Plant Material
Leaves of Buchholzia coriaceaEngl. (Caparridaceae) and Cogniauxia podolaena Baill (Cucurbitaceae) were collected in Brazzaville in 2017, and authenticated with the herbarium n˚ 2456 of 17 February 1968 collected by Bouquet; with the herbarium n˚ 548 of 4 August 1963 deposited by Neré. After harvesting, these leaves were cleaned and then dried for three (3) weeks at the Laboratory of Biochemistry and Pharmacology of the Faculty of Health Sciences in Brazzaville at an ambient temperature of 25˚C to 27˚C.
2.2. Animals Used
Virgin wistar rats aged 18 to 22 weeks with an average weight of 100 g were used to evaluate the uterotrophic, hormonal and biochemical activities of the aqueous extracts of the leaves of B. coriacea Engl. or C. podolaena. These animals came from the animal house of the Faculty of Science and Technology of the University Marien Ngouabi (Brazzaville-Congo). They were subjected to standard conditions of average temperature varying between 27˚C - 29˚C, photoperiod cycle of 12 hours of light/darkness. They received standard feed and tap water ad libitum.
2.3. Preparation of Plant Extracts
The aqueous extracts used in this study were prepared by decoction, a method of preparation used by most traditional healers: 100 g of pulverised dry leaves of B. coriacea or C. podolaena were boiled for 15 min in 1000 ml of distilled water. After filtration, the solution obtained was evaporated at 70˚C, using a Heidolph type heater for 48 h; the concentrate obtained was collected in powder form; and the yield was determined. Dissolving 1 g of the concentrated powder in 10 ml of distilled water gave a 100 mg/ml concentrated solution, which was used as an aqueous extract for administration to the rats.
2.4. Evaluation of Uterotrophic Activity of Aqueous Extracts of B. coriacea Engl. and C. podolaena Baill. Leaves in Castrated Rats
Uterotrophic activities were performed according to the method described by [14] [15] [16] [17] [18].
2.4.1. Ovariectomy in the Rat and Constitution of Batches for Testing
Bilateral ovariectomy was performed on each rat to assess the effect of each plant extract on the uterus and sex hormones in the absence of the ovaries.
Thus, five (5) uncastrated virgin rats and twenty (20) castrated virgin rats divided into five (5) batches of five (5) animals, received each morning during one week; respectively;
By mouth:
Distilled water (0.5 ml/100g), control for batch 1 (uncastrated rats),
Distilled water (0.5 ml/100g), control for batch 2 (castrated rats),
Cogniauxia podolaena extract (600 mg/kg), for batch 4 (castrated rats),
Buchholzia coriaceaextract (600 mg/kg), for batch 5 (castrated rats).
By subcutaneous route:
17-β-estradiol, (100 µg/kg), reference product for batch 3 (castrated rats).
2.4.2. Evaluation of the Live Weight of Rats Treated with Each Extract
The weight of ovariectomized and whole (cycled) rats treated with each extract was recorded daily using a precision balance and compared to control rats.
2.4.3. Assessment of Uterine Weight of Rats Treated with Each Extract
The ratio of fresh to dry uterine weights of the rats treated with each extract was determined to assess the activity of each extract on uterine weight.
Thus, (24 h) after the last administration of the products, the animals were sacrificed by cervical dislocation and the uterus carefully removed. The uterus was separated from the fatty adhesions and weighed using a Highland Adam precision balance with a capacity of 120 g and a sensitivity of 0.001 g to obtain the fresh weight of the uterus.
The uterus was then placed in an oven for 24 hours at 100˚C and reweighed to obtain the dry weight of the organ. The fresh and dry weights obtained were used to determine:
The change in the ratio of fresh to dry weight of the uterus in each batch;
The variation in fresh weight of the uterus between the different batches;
The variation of the dry weight of the uterus between the different batches.
2.4.4. Evaluation of the Chemical Composition of the Treated Rat Uterus
The 10% uterine homogenates were prepared by grinding the dry uterus in a Potter with 0.9% NaCl solution. The crushed solution was centrifuged at 4000 rpm for 60 min. The supernatant obtained after centrifugation was stored in the freezer at −4˚C; in 1.5 ml Eppendoff tubes for the determination of total cholesterol and protein in the uterus.
2.5. Evaluation of Sex Hormone Levels and Biochemical Parameters of Castrated Rats Treated with B. coriacea Engl. and C. podolaena Baill. Extracts
Hormonal and biochemical assays were carried out according to the methods described by [15] [19] [20]. Blood samples were taken at each stage of the sexual cycle; in the control and treated rats with each extract in order to compare the variation in sex hormone levels (oestrogen, progesterone) and biochemical parameters (proteins, total cholesterol) with respect to the controls.
Blood from each rat (castrated or not) was gently collected from the ophthalmic vein using Vitrex heparin tubes. After centrifugation of the blood at 4000 rpm for 30 min, the collected plasma was stored in a freezer at −4˚C in 1 ml Eppendoff tubes for the determination of hormones and biochemical parameters.
Vaginal smears using the Haris Shorr technique, described by [20], were only performed in cycled rats as ovariectomized rats have a blocked sexual cycle. Vaginal smears, blood sampling and administration of extracts were performed according to the scheme shown in Figure 1 below.
2.5.1. Techniques for the Determination of Oestradiol and Progesterone
The assays for oestradiol and progesterone were performed using the Cypress Diagnostics method. The Cypress Diagnostics Estradiol (or Progesterone) ELISA
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Figure 1. Diagram of vaginal smears, administration of extracts and blood sampling. DOs: di-œstrus; PrOs: pro-œstrus; Os: œstrus; PsOs: post-œstrus.
is an enzyme immunoassay for the quantitative determination of estradiol (or progesterone) in serum or plasma.
2.5.2. Blood and Uterine Cholesterol and Protein Assays
1) Serum and uterine total cholesterol assay
This assay is based on an enzymatic method described by Allain et al. (1974) [21] by Bayala (2005) [15]. A drop of the sample (blood or homogenate) is placed on the plate and then spread evenly through the spreading layer into the underlying layers. Read the absorbance of the sample and standard against the reagent blank at 546 nm within 60 min of the end of the incubation.
2) Determination of total serum and uterine proteins
Protein determination was performed according to the method of Gornall, described by [22]. In a basic medium, sodium and potassium tartrate form a soluble complex with cupric ions. The addition of a protein displaces the copper complexed with the tartrate to form a violet-coloured copper-protein complex which shows an absorption maximum at 540 nm. The concentration of the protein to be assayed is then deduced.
3. Results
3.1. Uterotrophic Activity of B. coriacea (BC) Engl. and C. podolaena (CP) Baill
3.1.1. On the Live Weight of the Rats
Figure 2 shows the weight evolution of the rats during the trial in the 5 batches: (RnC + ED, RC + ED, RC + E2, RC + BC and RC + CP). It shows that the castrated control rats showed a greater weight change compared to the non-castrated control rats (p < 0.05). The administration of aqueous extracts of B. coriacea and C. podolaena at 600 mg/kg to castrated rats did not result in any difference in
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Figure 2. Evolution of live weight of castrated rats treated with Buchholzia coriacea and Cogniauxia podolaena extract at 600 mg/kg (n = 4). RnC + ED: Uncastrated rats + distilled water; RC + ED: Castrated rats + distilled water; RC + BC: Castrated rats + Buchholzia coriacea; RC + CP: Castrated rats + Cogniauxia podolaena; RC + E2: Castrated rats + 17β-estradiol; *: Significant difference; ns: Not significant difference.
weight development compared to control castrated rats (p > 0.05). In contrast, 17-β-estradiol administered at 100 µg/kg resulted in a significant decrease in the live weight of castrated rats compared to control castrated rats and those treated with each extract (p < 0.05).
3.1.2. On Fresh and Dry Weight of the Uterus
Figure 3 and Figure 4 show a fresh and dried uterus respectively. These figures show the significant decrease in size and weight when a uterus is changed from fresh to dry.
Table 1 shows the average fresh uterus weight, the average dry uterus weight and the ratio of fresh to dry uterus weight in each of the 5 batches. It shows that ovariectomy of the rats causes a significant decrease in the ratio of fresh to dry weight of the uterus. Administration of aqueous extract of BC or CP at 600 mg/kg in castrated rats does not cause a change in this ratio. However, administration of 17β-estradiol at 100 µg/kg significantly increased the ratio in castrated rats.
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Figure 3. Photograph of a fresh uterus of a castrated rat.
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Figure 4. Photograph of a dry uterus of a castrated rat.
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Table 1. Fresh weight to dry weight ratio of rat uteri under the effect of aqueous extracts of Buchholzia coriacea and Cogniauxia podolaena (600 mg/kg).
Values are means ± MSE with n = 4. o: non-significant difference compared to uncastrated rats of the control lot; a: significant difference compared to uncastrated rats of the control lot; *: (p < 0.05), significant difference compared to castrated rats of the control lot; ns: non-significant difference compared to castrated rats of the control lot; E2: estradiol; ED: distilled water; BC: Buchholzia coriacea; CP: Cogniauxia podolaena; RnC: uncastrated rats; RC: castrated rats; P. fresh/dry: fresh and dry weight.
3.2. Uterine Levels of Total Cholesterol and Protein
The uterine concentrations of total cholesterol and protein of the rats after 6 days of treatment and subsequent sacrifice of the animals are presented in Table 2. It indicates that after ovariectomy, uterine protein levels decrease and cholesterol levels increase compared to uncastrated control rats (p < 0.05). Administration of aqueous extract of BC or CP at 600 mg/kg, does not cause significant changes in the level of the two biochemical parameters in the uterus of castrated rats. However, 17β-estradiol promotes protein increase and cholesterol decreased in the uterus of castrated rats.
3.3. Sex Hormone Levels and Biochemical Parameters of Castrated RATS Treated with B. coriacea Engl. and C. podolaena Baill
The blood concentrations of estradiol, progesterone, cholesterol and proteins of the rats according to the phases of the sexual cycle during the test are presented in Table 3.
3.3.1. Estradiol and Progesterone Levels
Table 3 shows that in uncastrated control rats, the mean concentration of estradiol and progesterone in the blood varies with the estrous stage: it is low in di-estrus (10.91 ± 3.20 pg/l and 3.08 ± 0.65 ng/l), increases in pro-estrus (141.60 ± 15.50 pg/l and 4.04 ± 0.21 ng/l), and peaks in the estrus stage (608.60 ± 209 pg/l and 30.50 ± 4.12), before decreasing in post-estrus (103.60 ± 12.10 pg/l and 4.51 ± 0.22 ng/l).
After ovariectomy, the peak levels of estradiol and progesterone decreased significantly in the blood of control castrated rats. Administration of aqueous extract of BC or CP at 600 mg/kg in castrated rats does not result in changes in estradiol or progesterone levels compared to control castrated rats (p > 0.05).
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Table 2. Protein and total cholesterol levels in the uterus of castrated rats treated with Buchholzia coriaceaand Cogniauxia podolaena extract at 600 mg/kg.
Values are means ± MSE with n = 4. o: non-significant difference compared to uncastrated rats of the control lot; a: significant difference compared to uncastrated rats of the control lot *: p < 0.05), significant difference compared to castrated rats of the control lot; ns: non-significant difference compared to castrated rats of the control lot E2: estradiol; ED: distilled water; BC: Buchholzia coriacea; CP: Cogniauxia podolaena; RC: castrated rats. RnC: non-castrated rats. In bold significant values.
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Table 3. Blood levels of estradiol, progesterone, cholesterol and protein in castrated and non-castrated rats treated with Buchholzia coriaceaand Cogniauxia podolaena extracts at 600 mg/kg (n = 5).
Values are expressed as means (M) ± standard error of the mean (SEM); n = 4. 0: non-significant difference between di-estrus and estrus stages of uncastrated rats; 1 and 2: significant and highly significant difference between di-estrus and estrus stages of uncastrated rats. o: non-significant difference compared to uncastrated rats in the control lot; a and b: significant and highly significant difference compared to uncastrated rats in the control lot. ns: non-significant difference compared to castrated rats in the control group. *: (p < 0.05) and **: (p < 0.01): significant and highly significant difference compared to castrated rats of the control lot. E2: 17β estradiol; P4: progesterone; ED: distilled water; BC: Buchholzia coriacea; CP: Cogniauxia podolaena; Prot: protein; Chol: total cholesterol; RnC: non-castrated rats; RC: castrated rats. In bold are significant values.
However, administration of 17β-estradiol to castrated rats resulted in a significant increase in estradiol and progesterone levels compared to control castrated rats (p < 0.05).
3.3.2. Total Cholesterol and Protein Levels
The same Table 3 shows that the administration of the aqueous extract of BC or CP at 600 mg/kg in castrated rats did not cause any change in total cholesterol level (p > 0.05), but each extract caused a significant increase in protein level (p < 0.05) as with the rats treated with 17β-estradiol, the reference product.
4. Discussion
The present study showed than aqueous extract of the leaves of Buchholzia coriacea (BC) Engl. or Cogniauxia podolaena (CP) Baill. administered at a dose of 600 mg/kg for one week in castrate d rats did not induce uterotrophic effects, nor did it cause significant variations in the level of sex hormones. However, a significant increase in blood protein levels was observed with both plant extracts.
Ovariectomy of the rats caused a significant increase live weight of the rats compared to uncastrated control rats during the trial period. Administration of 17β-estradiol (E2) to castrated rats significantly decreased this weight compared to control castrated rats that received distilled water. This result is in agreement with those of [23] who reported that, when ovariectomizing female rats, the loss of estrogen production due to the absence of the ovaries caused overfeeding and led to rapid weight gain. [24] reported that in castrated rats, 17β-estradiol facilitates lipolysis and causes weight loss; thus estrogen deprivation leads to obesity and estrogen therapy counteracts this, exerting anti-lipogenetic effects. In this study, 17β-estradiol, by reducing the weight of castrated rats, allowed some restoration of ovarian function after oophorectomy.
However, administration of the aqueous extracts of BC and CP to castrated rats did not cause a significant change in body weight of the castrated rats compared to control rats that received distilled water. The lack of effect of both extracts on body weight in ovariectomized rats suggests that both extracts did not bind to estrogen receptors as with 17β estradiol. Indeed according to [25] [26] [27], 17β estradiol, estrogen and phytoestrogens bind to estrogen receptors, they mimic some of their effects by interaction with ERα and Erβ receptors, even in the absence of the ovaries. Noting that in whole uncastrated or cycled rats, B. coriacea caused the decrease in weight and C. podolaena caused the increase [12]. These observations suggest that the effect of both extracts on rat weight requires the presence of ovaries, whereas the effect of 17β estradiol on rat weight did not require the presence of ovaries. These extracts are assumed not to have the same mechanism of action as the reference molecule 17β-estradiol, which implies that they would not contain plant hormones or phytohormones.
In addition, administration of the aqueous leaf extract of both plants did not cause a change in the fresh to dry weight ratio of the uterus, an indicator of the level of estrogenic impregnation of the uterus, whereas 17β-estradiol increased it. The determination of estradiol (E2) and progesterone (P4) levels in this study showed that in uncastrated or cycled control rats, the level of each sex hormone is higher at the estrus stage and lower at the di-estrus stage. This result is in agreement with those reported by [28] and [29] who indicated that in mammals, maximum levels of estradiol and progesterone are observed at the end of proestrus and the beginning of estrus, and minimum levels at di-estrus. Indeed, during the ovulatory phase in women, estradiol levels vary considerably between 30 and 400 pg/ml and progesterone levels between 2 and 25 ng/ml [30] [31]. These values are comparable to the large variations in E2 or P4 levels observed in this study during the estrus phase in the same batch of rats. After ovariectomy, E2 and P4 levels dropped significantly in all rats during the assay period. This indicates that the sexual cycle of castrated rats was effectively blocked.
Administration of B. coriacea and C. podolaena did not cause significant changes in sex hormone levels (estrogen and progesterone) in castrated rats, whereas 17β-estradiol did. This observation corroborates the hypothesis of the absence of plant hormones or phytohormones in the two extracts studied. Indeed [32] reported that the administration of 17β estradiol, a natural hormone at physiological doses in ovariectomized mice increased the number of estrogen receptors, i.e., the increase in circulating estrogen levels. In addition, both extracts showed no effect on uterine protein or cholesterol levels in castrated rats, however rats treated with 17β-estradiol showed an increase in uterine protein levels and a decrease in uterine total cholesterol levels. According to [15] the increase in uterine protein by estradiol or phytohormones is explained by uterine cell proliferation and the decrease in uterine cholesterol is explained by its use in the process of steroidogenesis. We therefore believe that these extracts would not contain the plant hormones, as they do not provide hormone supplementation, but may be use from hormone phytomodulators.
Blood protein levels increased significantly in rats treated with each plant extract, as with 17β estradiol. This assumes that while uterine protein levels depend on ovarian function, blood protein levels appear to be independent of this function. This study opens up the prospect of deepening the mechanisms of action of phytomodulating plants of hormones and biochemical parameters.
5. Conclusion
The aqueous extracts of the two plants did not show any uterotrophic and hormonal effects in the castrated rat; their estrogenic and progesteronic effects observed in the whole or cycled rat would be ovary-dependent, so they would not contain phytohormones. Both plants could be used as hormone phytomodulators, but their use by traditional medicine in the treatment of hormone deficiencies is not justified.