Background

Periploca is a genus of plants from the Asclepiadaceae family in the major group of angiosperms. Several species of this family, such as P. angustifolia are widely used in traditional medicine as anti-diabetic, anti-mutagenic, and anti-rheumatic agents [1]. In Egypt, the leaves of P. angustifolia are used to treat rheumatic diseases and the roots are used for hemorrhoids, gastric ulcer and diabetes. Its resin is used as a hypotensive [2] a masticator when burning. P. angustifolia L was used by the Bedouins as animal food and herbal remedies [3]. Different plant extracts of the Asclepiadaceae family have shown significant anti-inflammatory [4] and anti-ulcerogenic properties [5]. In addition, the methanolic extract of P. angustifolia leaves has been shown to have antioxidant effects and exert antidotal effects on cadmium-induced hepatotoxicity [6]. There is a relationship between the antioxidant capacity and anti-hyperglycemic potential of Periploca sylvestre and it may be due to flavonoids and phenolic contents in the plant that impart these properties [7]. Gymnema sylvestre (Asclepiadaceae) has been used since ancient times as a folk medicine for the treatment of diabetes, obesity, and stomach stimulation uses [8]. Aerial parts of P. angustifolia L. collected from southern Tunisia possess antioxidant activity against 2, 2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid (ABTS) and 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) [9]. Samples of Hemidesmus indicus var. indicus and var. pubescens during the flowering season possess higher antiulcer and anti- hepato-carcinogenic effects [10, 11]. The chemical composition of the root bark of P. angustifolia, at the flowering stage showed the presence of C-heterosids (anthracenic derivatives), anthocyans, saponins, free quinons and proanthocyanidols [12]. In this study we determined the phytochemical composition and, in particular the phenolic and flavonoids composition of the ethanolic extract of P. angustifolia L., and we assessed anti-inflammatory, anti-ulcerogenic, and hypoglycemic activities.

Methods

Plant material

P. angustifolia L. was collected from the Sallum Plateau (northwestern coast of Egypt) during 2012–2013. The plants were air dried at lab-temperature until their weight plateaued, and then ground to a fine powder. The different parts of the plants were identified, confirmed and authenticated by comparing with an authentic specimen at the Plant Taxonomy Unit, Desert Research Center, Cairo, Egypt. The samples were extracted by percolation in ethanol 70%, filtered and this step was repeated several times. The ethanolic extract was concentrated under reduced pressure at temperatures not exceeding 40 °C. The obtained ethanol extract of P. angustifolia L. constituted 10% from the entire dried plant and was used for subsequent investigations. Scheme of separation for flavonoids and phenolics from the whole plant of P. angustifolia L was illustrated in Fig. 1.

Fig. 1
figure 1

Scheme of Separation of flavonoids and phenolics from the whole plant of P. angustifolia L

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Drugs

Diclofenac sodium (Voltarin®) was obtained from Novartis Pharma Co. (Cairo, Egypt) under license from Novartis Pharma AG, (Basle, Switzerland). Ranitidine hydrochloride tablets (Zantac® Batch No. 001716C) were manufactured by Glaxo-Wellcome Egypt (Elsalam City, Cairo, Egypt, each tablet contained 150 mg ranitidine). Glibenclamide (Daonil®) was purchased from Aventis Co., under license from Aventis Pharma Co., West Germany.

Animals

Wistar Albino rats (150–170 g) and Swiss mice (18–22 g) were obtained from the Laboratory Animal Colony, Helwan, Egypt. Animals were maintained in the Animal House of the Pharmacology Department (Faculty of Veterinary Medicine, Cairo University) under controlled conditions [temperature 23 ± 2 °C, humidity 50 ± 5% and 12-h light-dark cycles]. All animals were acclimatized for 7 days before the study. The animals were housed in sanitized polypropylene cages, containing sterile paddy husk as bedding. Animals were habituated to laboratory conditions for 48 h prior to the experimental protocol to minimize non-specific stress. All animals were fed a balanced diet of wheat bran, soybean powder, fish-meal and dietary fibers (manufactured by Cairo Agricultural Development Co.). Water was provided ad libitum. The Institutional Animal Care and Use Committee (IACUC), Cairo University approved this study.

Estimation of Total flavonoids

The flavonoid content in extract was determined spectrophotometrically according to the method described by Djeridane et al. [13], which is based on the formation of a flavonoid–aluminum complex with a maximum absorbance at 430 nm. Total flavonoids are expressed as mg quercetin equivalent.

Estimation of Total phenolic content (TPC)

The Folin-Ciocalteu method [14] was used to determine the TPC spectrophotometrically in the different extracts using gallic acid as standard. The TPC was expressed as μg/mg gallic acid equivalent (GAE).

Identification of phenolic and flavonoids

HPLC was used to identify phenolics and flavonoids. A known weight of air-dried plant powder was soaked in 25 ml sterilized water and agitated on a rotary shaker for 24 h at 200 rpm. Slurry was filtered through Whatmann 3MM filter paper under a vacuum, followed by centrifugation at 12.5 rpm for 30 min at 80 °C. The aqueous extract was acidified to pH 2.5 using diluted phosphoric acid. The sample was sacked three times through a separating funnel with an equal volume of diethyl ether. The combined diethyl ether layer was evaporated to dryness under reduced pressure at 30 °C. The resulting residue was re-dissolved in 3 ml of HPLC-grade methanol and filtered through a sterile membrane with a pore size of a 0.2 μm prior to HPLC analysis [15]. Identification of individual phenolic compounds of the plant sample was performed using a Dionex (Model 3000) HPLC, using a BDS Hypersil C18 reversed-phase column (250 × 4.6 mm) with 10 μm particle size. Injection by means of Rheodyne injection valve (Model 7125) with a 50 μl fixed loop. A constant flow rate of 1 ml/min was used with two mobile phases: distilled water (A), and acetonitrile (B), using a UV detector set at wavelength 254 nm. Phenolic compounds of each sample were identified by comparing their relative retention times with those of the standard mixture chromatogram. The concentration of an individual compound was calculated on the basis of peak area measurements, and then converted to μg/g phenolic dry weight. All chemicals and solvents used were HPLC spectral grade. Standards phenolic compounds were obtained from Sigma (St. Louis, USA) and Merck (Munich, Germany).

Acute toxicity

The acute toxicity (LD50) of ethanolic extract of P. angustifolia administered orally was estimated in mice using Lorke [16] method. Three groups of five animals received 10, 100, 1000 mg/kg of the extract suspended in Tween80 (vehicle 3% v/v). The animals were observed for 72 h for signs of toxicity and death. When no deaths were recorded another four groups of five mice were administrated 2000, 3000, 4000 and 5000 mg/kg of the extract orally. The animals were observed for 72 h for signs of toxicity and the number of deaths was recorded. Control animals were received the equivalent volume of vehicle. The LD50 values were calculated as the geometric mean of the highest non-lethal and the lowest lethal doses mathematically according to the Kerber method [17] using the following formula:

$$ \mathrm{LD}50=\mathrm{LD}100-\Sigma\ \left(\mathrm{z}\ \mathrm{x}\ \mathrm{d}\right)/\mathrm{m} $$

where z is half of the sum animals that died with the two next doses; d is the interval between two next doses and m is the number of animals/group.

Anti-inflammatory activity

The extract was evaluated for its anti-inflammatory activity in rats using the formaldehyde-induced paw edema method [18]. Acute inflammation was produced by sub-plantar injection of 0.2 ml formaldehyde (1% w/v) into the hind paw 1 h after oral administration of ethanolic extract of P. angustifolia (500 mg/kg b.wt.) or diclofenac sodium (50 mg/kg b.wt.) as a standard anti-inflammatory agent. The paw volume was measured in mm by a plethysmometer (Ugo-Basile, Italy) at 1, 2, 3, and 4 h after the formaldehyde injection. Inhibition of inflammation was calculated using the following formula: % inhibition = 100 (1-Vt/Vc), where ‘Vc’ represents edema volume in the control group and ‘Vt’ represents edema volume in the test group.

Anti-ulcerogenic activity

All rats were fasted for 48 h but were given water ad libitum till the start of the experiment. To prevent excessive dehydration during the fasting period, rats were supplied with sucrose (BDH) 8% (w/v) solution in NaCl (BDH) 0.2% (w/v), which was removed 1 h before experiments [19]. The animals were randomly separated into three groups of six rats. One group was pretreated with ethanolic extract of P. angustifolia orally at 500 mg/kg b.wt., another group received ranitidine (100 mg/kg orally) and the control group received equivalent volumes of saline instead of plant extract. One hour later, all groups were treated with ethanol (50%) at a dose of 10 ml/kg. One hour after ethanol administration, all rats were euthanized by an overdose of chloroform and the abdomen was opened. The stomach was removed, opened along the greater curvature, and gently rinsed under running water. The tissues were fixed with 10% formaldehyde in saline. Macroscopic examination was carried out under a hand lens and the presence of ulcer lesions was scored [20]. Lesions in the glandular part of the stomach were measured under an illuminated magnifying microscope (10 x). Long lesions were counted and measured along their greater length. Petechial lesions were counted with the aid of 1-mm squares grid [21]. Each five petechial lesions were considered to represent a 1-mm ulcer. The ulcer index (%) for each group was calculated as the sum of the lengths of long ulcers and petechial lesions divided by its number.

Hypoglycemic effect of P. angustifolia L. extract

Induction of diabetes

Rats were rendered diabetic by subcutaneous injection of alloxan monohydrate (Oxford) at a dose of 150 mg/kg/day for 3 days (early ketosis) and normal feeding was maintained [22]. Five days later, blood samples were drawn and the blood glucose level was measured to establish the occurrence of diabetes. The threshold for diabetes in the present study was a glucose level of ≥225 mg/dl.

Effect of P. angustifolia L. extract on hyperglycemic rats

The hypoglycemic effect of P. angustifolia L. extract was studied in alloxan-induced diabetic rats. The animals were fasted for 8 h but allowed free access to water. The diabetic animals were randomly divided into three groups of 10 rats and received oral P. angustifolia L. extract (500 mg/kg b.wt.), glibenclamide (0.2 mg/kg) or 20% v/v Tween 80 (5 ml/kg b.wt.).

Effect of P. angustifolia L. extract on Normoglycemic rats

Non-diabetic rats were fasted overnight and then, randomly divided into three groups of 10 rats. As with the diabetic rats, the non-diabetic received oral P. angustifolia L. extract (500 mg/kg b.wt.), glibenclamide (0.2 mg/kg b.wt.) or 20% v/v Tween 80 (5 ml/kg b.wt.). One milliliter of blood was collected before and after two hours of treatments from each rat in all groups. Blood glucose was estimated by the glucose oxidase method using the Randox kit (Randox Laboratories Ltd., Ardmore, UK) according to the manufacturer’s instructions.

Statistical analysis

The results are expressed as the mean ± standard error (SE). The differences between the experimental groups were analyzed by one-way analysis of variance (ANOVA), followed by Bonferroni’s test using SPSS 16.0 (SPSS Inc., Chicago, IL, USA). The results were considered statistically significant when p values less than 0.05 and 0.01 were considered significant (*p < 0.05, **p < 0.01).

Results

Acute toxicity

There were no changes in the general behavior of the animals at any dose and, there were no deaths after 72 h at the highest administered dose (5000 mg/kg) of the extract. The safety of the extract was shown by the high LD50 value of the extract (> 5 g/kg).

Total flavonoids and phenolic contents

The total flavonoids and phenolic contents of P. angustifolia L were 3.15 ± 0.7% as quercetin equivalent and 2.69 ± 0.6% as gallic acid equivalent, respectively.

Identification and quantification of phenolic and flavonoid compounds

Quantitative and qualitative analysis of the phenolic and flavonoid compounds in the ethanolic extract of P. angustifolia L. was performed using HPLC, where each compound was separated and identified using authentic standard. The compounds were, coumarin, resorcinol, isorhamnetin, quercetin, and naphthalene with different concentration ranges (Table 1 and Fig. 2). Resorcinol reached its maximum value of 56.54% in ethanolic extract of P. angustifolia L. followed by isorhamnetin at 40.53%, while quercetin was estimated to have a minimum value of 0.002%.

Table 1 Phenolic and flavonoid compounds identified in the ethanolic extract of P. angustifolia L. using HPLC
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Fig. 2
figure 2

HPLC chromatogram of the phenolic and flavonoid compounds of the P. angustifolia L

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Anti-inflammatory activity

The formaldehyde-induced paw edema model showed that sub-plantar injection of formaldehyde in rats caused a time-dependent increase in paw thickness and the maximal increase was observed 4 h after formaldehyde administration (Table 2). However, rats that received the ethanolic extract of P. angustifolia pretreatment showed significantly less (P < 0.05, P < 0.01) formaldehyde-induced inflammation at each time point than the animals that received formaldehyde only as did those that received the reference anti-inflammatory drug diclofenac sodium (P < 0.01). Oral administration of P. angustifolia L. extract at 500 mg/kg resulted in a maximal inhibition of paw inflammation 42.42% that was close to that of diclofenac sodium 45.45% at 50 mg/kg 4 h post formaldehyde administration (Fig. 3).

Table 2 Formaldehyde induced paw edema measured as paw thickness in rats treated with P. angustifolia L. extract and in control rats (n = 5)
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Fig. 3
figure 3

Anti-inflammatory activity of Periploca angustifolia L. extract by formaldehyde induced rat paw edema (n = 5)

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Anti-ulcer activity

Ethanol caused extensive gastric damage in the mucosa of the control animals. The lesions were characterized by multiple long hemorrhagic red bands of different sizes along the axis of the glandular stomach with petechial patches. By contrast, oral treatment with the ethanolic extract of P. angustifolia L showed significantly fewer (p < 0.01) the gastric lesions and lower ulcer index than those observed in the control animals. The crude ethanolic extract of P. angustifolia L. had a protective index of 44.93%, whereas ranitidine as a reference standard (100 mg/kg) exhibited a protective index of 46.99% indicating a potent anti-ulcerogenic effect of P. angustifolia L. extract (Table 3 and Fig. 4).

Table 3 The effect of P. angustifolia L. extract on alcohol-induce ulcer in rats (n = 5)
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Fig. 4
figure 4

The anti-ulcerogenic effect of Periploca angustifolia L. on Alcohol -induce ulcer in rats (n = 5)

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Hypoglycemic activity

Subcutaneous injection of rats with alloxan resulted in a significant increase in serum glucose levels. Administration of the crude ethanolic extract of P. angustifolia significantly reduced blood glucose levels at 2 h compared with those untreated diabetic rats. Specifically, the ethanolic extract significantly reduced the postprandial blood glucose level of diabetic rats from 211.16 to 124.33 mg/dl (Table 4 and Fig. 5). Similarly, the extract of P. angustifolia at the same dose significantly reduced blood glucose level in normoglycemic rats (Table 5 and Fig. 6).

Table 4 Hypoglycemic activity of P. angustifolia L. extract in alloxan-diabetic rats (n = 10)
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Fig. 5
figure 5

Hypoglycemic activity of Periploca angustifolia L extract in alloxan-diabetic rats (n = 10)

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Table 5 Hypoglycemic activity of P. angustifolia L. extract in non-diabetic rats (n = 10)
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Fig. 6
figure 6

Hypoglycemic activity of Periploca angustifolia L extract in non-diabetic rats (n = 10)

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Discussion

P. angustifolia extract had a high safety margin in mice as the LD50 was > 5 g/kg. Similarly, Sunil et al. [23] found that alcoholic extract of another plant in the Asclepiadaceae family Holostemma ada kodien was non toxic at a dose of 5 g/kg b.wt.

Phenolic compounds increase a plant biological value because they exhibit a range of pharmacological properties, such as anti-diabetic, anti-allergenic, anti-atherogenic, anti-inflammatory, antioxidant, anti-thrombotic, and vasodilator effects [24, 25]. Oxidative stress activates inflammatory pathways in stem cells and progenitor cells, leading to exhaustion of these cells due to increased levels of reactive oxygen species (ROS) [26]. Cellular exhaustion in turn leads to the development of several diseases, such as gastrointestinal ulcers, hyperglycemia, and hepatic dysfunction [27]. Thus, natural antioxidants provide cellular protection and lead to favorable effects in diabetes mellitus [28] and the majority of inflammatory and cardiovascular diseases [29]. Examples of naturally occurring antioxidants are flavonoids, phenolic acids, coumarin, isorhamnetin and quercetin that were separated and identified from P. angustifolia extract with promising anti-inflammatory agents. This activity may be due to their inhibitory action on neutrophils infiltration, cyclooxygenase-2 activity and inflammatory cytokines release [30,31,32]. The anti-ulcerogenic activity of P. angustifolia L. may also be due to the presence of quercetin, as it prevents gastric mucosal damage by increasing mucus production with a comparable regression of gastric lesions [33]. Hyperglycemia is a metabolic disorder that occurs due to the excess production of ROS, which destroy pancreatic β-cells and is associated with vascular complications including neuropathy, retinopathy, and nephropathy [34]. Herbal medicines have long been used for the treatment of diabetes mellitus and have fewer side-effects of toxicity than other hypoglycemic drugs. In the present study, administration of the ethanolic extract of P. angustifolia significantly reduced blood glucose levels in normoglycemic and diabetic rats. This promising effect may be attributed to the inhibition of aldose reductase which converts glucose to sorbitol and α-amylase and α-glucosidase (key enzymes linked to type-2 diabetes) by the phenols and flavonoids [35, 36].

Because of its basic chemical structure, quercetin is a antioxidant activity and it is now used as a nutritional supplement for a variety of diseases such as diabetes/obesity and circulatory dysfunction, including inflammation as well as mood disorders [37].

Conclusion

The crude ethanolic extract of P. angustifolia exhibited promising anti-inflammatory, anti-ulcerogenic, and hypoglycemic activities, which are in accordance with its use folk medicine. As the extract showed a high, safety profile this study may serve as a guideline for the standardization and validation of natural drugs containing selected medicinal plants ingredients. Moreover, further investigation for the selective activities is required to determine the exact mechanism of action.