PHARMACOLOGICAL AND BIOLOGICAL EVALUATION OF METHANOL EXTRACTS OF SELECTED CHOLISTANI PLANTS
F. Sadiq1, H. Habib2, M. I. Shahzad1*, K. Fatima2, H. Saeed2, M. Shafiq2, S. Ajmal2, T. Noor2, M. Mustafa3, H. Ashraf2, I. Saeed4 S. Hameed5 and M. Abdullah6
1 Department of Biochemistry, Institute of Biochemistry, Biotechnology and Bioinformatics, The Islamia University of Bahawalpur, Bahawalpur-63100, Pakistan.
2 Department of Botany Government Sadiq College Women University, Bahawalpur-63100, Pakistan.
3 Department of Biotechnology, Institute of Biochemistry, Biotechnology and Bioinformatics, The Islamia University of Bahawalpur, Bahawalpur-63100, Pakistan.
4 Department of Chemistry, The Islamia University of Bahawalpur, Bahawalpur-63100, Pakistan.
5 Department of Anatomy, Faculty of Veterinary and Animal sciences, The Islamia University of Bahawalpur, Bahawalpur-63100, Pakistan.
6 Cholistan Institute of Desert studies, The Islamia University of Bahawalpur, Bahawalpur-63100, Pakistan.
Corresponding Author email: mirza.imran@iub.edu.pk
F. Sadiq and H. Habib both are first authors
ABSTRACT
Cholistani plants are a rich source of bioactive compounds that are helpful in treating various ailments. The objective of the current investigation was to elucidate the pharmacological importance of four Cholistani plants including L. indicum, E. granulata, C. prostratus, and H. crispum. Methanolic extracts of these plants were subjected to antibacterial, antibiofilm, antioxidant, antidiabetic, and antiviral analysis. Disc diffusion assay was used for antibacterial activity and the MIC of active extracts was also calculated. In vitro antibiofilm assay was conducted against drug-resistant and drug-sensitive bacterial strains and % inhibition was calculated. DPPH assay was used for the evaluation of antioxidant potential. The antidiabetic potential was tested by α-glucosidase inhibitory assay. The Haemagglutination (HA) test was performed to assess the antiviral properties of these plants against the Avian Influenza Virus (H9N2). It was found that E. granulata prevailed in antibacterial potential with max ZoI against selected bacterial strains (16.5 mm versus E. coli. C. prostratus) surpassed all other plant extracts in terms of biofilm inhibition with up to 90% inhibition against P. aeruginosa. Considerable antioxidant potential was revealed by all examined plants in the order of E. granulata> C. prostratus > L. indicum > H. crispum. A substantial α-glucosidase inhibitory potential was detected in all the studied plants. All of the examined plants displayed significant (titer 0) antiviral activity. This study reveals that all of these plants have the potential to be employed as antibacterial, antioxidant, and antiviral agents. In vitro suppression of the α-glucosidase enzyme indicated that these plants are potent sources of antidiabetic compounds.In conclusion, all of the examined Cholistani plants are rich sources of pharmacological compounds and should be further researched for drug development.
Key words: E. granulata, C. prostratus, L. indicum, H. crispum, antibacterial, antioxidant, antibiofilm, antiviral:
INTRODUCTION
The term ‘Cholistan is derived from the Turkish word ‘Chol’ and this signifies the ‘land of the desert’. It is also called the ‘Rohi desert’ that surrounds an area of 30km from Bahawalpur (Ahmad et al., 2012). The usage of medicinal plants as medications has dramatically grown in recent years. According to the global health organization, 65 to 80% of humans rely on plants for healing ailments owing to the expensive cost and adverse side effects of synthetic pharmaceuticals (Mesfin et al., 2013). Various components of plants such as seeds, roots, berries, leaves, bark, and flowers are employed by common practitioners for health care ever since ancient times (Mustafa et al., 2017).
L. indicum, a medicinal herb from the Cholistan desert, belongs to the Aizoaceae family and is known as "Baluka sag" and "jungli lonak" (Wariss et al., 2013). Euphorbia granulata Forssk, also known as Hazar Dani, is a prostrate, annual plant found worldwide (Webster, 2014). Heliotropium crispum, also known as kali Buri, is a medicinal desert plant used in herbal treatments and is fed to cattle to promote lactation (Qureshi et al., 2010). Convolvulus prostratus Forssk, also known as "Hiran Booti," belongs to the morning glory family and is found in tropical, subtropical, and temperate climates (Ekka and Dixit, 2007).
Plants contain phytochemicals like terpenoids, tannins, alkaloids, and flavonoids that have antibacterial properties (Bhalodia and Shukla, 2011). As antibiotic resistance and side effects increase, research on plants' antimicrobial role against resistant strains is gaining traction, as they are considered safe and efficacious (Algabr et al., 2022). Biofilm formation on medical devices poses significant challenges in medical research, particularly in urology, where it is a primary source of urogenital infections (Tenke et al., 2012). Biofilm-associated infections are difficult to cure, even in people with robust immune systems, as bacteria in biofilms may evade host defense. This has led to a search for new antimicrobial chemicals (Teanpaisan et al., 2017). A substance that considerably reduces or delays oxidative stress is known as an antioxidant molecule. Free radicles cause substantial oxidative damage to the body and are involved in causing various ailments (Venkatachalam et al., 2012). The modern world is concentrating on receiving antioxidants from natural sources as a health supplement (Kumar et al., 2022).
An antioxidant molecule reduces or delays oxidative stress, which can cause various ailments. In type 2 diabetes, inhibiting the activity of glucosidase enzymes may lower hyperglycemia. Diabetic drugs that inhibit these enzymes slow down starch digestion in the small intestine, resulting in less glucose production and circulation (Briones and Bajaj, 2006). Commercial drugs for suppressing α-glucosidase have adverse effects and high costs (Adefegha and Oboh, 2012). Research is being conducted to develop innovative anti-glucosidase and anti-diabetic medicines with enhanced safety profiles for long-term treatment (Liu and Ma, 2017). The highly pathogenic avian influenza (HPAL) virus causes severe illness in domestic poultry, particularly H9N2, which is associated with morbidity in Pakistan and South East Asia (Wu et al., 2016). It is believed to be causing a new influenza pandemic in humans in the region. Herbal medications and plant extracts have shown strong antiviral benefits (Verma et al., 2008). Keeping in mind these challenges, present investigation was carried out to assess the antimicrobial, antibacterial, antioxidant, anti-viral, and anti-diabetic potentials of selected Cholistani plants.
MATERIALS AND METHODS
Specimen Collection: Selected plants were collected from herbarium of the Cholistan Institute of Desert Studies (CIDS), The Islamia University of Bahawalpur, Pakistan. Study was conducted from April 2021 till August 2021. Plant identification was done by taxonomist at CIDS and voucher numbers were issued to plants. Limeum indicum CIDS/IUB-0205/7, Heliotropium crispum CIDS/IUB-0502/23, Convolvulus prostratus CIDS/IUB-1103/46 and Euphorbia granulata CIDS/IUB-1602/60-B.
Extract preparation: The whole plants were collected, cleaned, and dried under shade for 10 days. The dried plants were converted into powder form and 10 g of each plant powder was dissolved in 100 ml of Methanol (MtOH) in an airtight container. The solution was kept at 37°C under constant shaking for 96 hrs. Later the samples were filtered through Whatman filter paper No. 1. The filtrates were subjected to a rotary evaporator at 45-50°C and after complete removal of solvent, the precipitates were collected, weighed, and dissolved in MtOH @ 500 mg/mL and 10 mg/mL (Stanković et al., 2016).
Antibacterial activity: Disc diffusion assay was performed for antibacterial evaluation against selected bacterial strains.The pre-soaked GF-1 grade filter paper discs of extracts and control were used. The bacteria were inoculated on agar plates, incubated for 40 min at 37 °C, and then discs were applied. The culture was again incubated at 37 °C overnight and the zone of inhibition (ZoI) was recorded by the standard method by CLSI guidelines. The pure solvent was used as the negative control. Ampicillin and Moxifloxacin were used as a positive control for normal and drug-resistant bacteria,respectively (Jahan et al., 2018).
Minimum inhibitory concentrations (MIC): MIC was determined using the broth-dilution technique according to the protocol given by Wiegand et al., (2008) with slight modifications. 50 µl of nutrient broth and 50 µl of fresh bacterial culture were added to wells of sterilized microtiter plate. 100 µl of plant extract and positive control drug were added to first well and serially diluted till 11th well. 12th well was used as negative control. Plate was incubated at 37°C overnight. Next day INT (Iodonitrotetrazolium chloride) (20 µl) was added to each well and plate was incubated at 37°C for 20-30min and color change was observed.
Antibiofilm assay: The antibiofilm potential of selected plant extracts was evaluated by the standard method given by Bazargani and Rohloff, (2016). OD630nm was taken through an ELISA reader and % inhibition was calculated by the formula (%Inhibition = [(A0-AX)/A0] *100), where A0 is the absorbance of negative control and AX is the absorbance of the sample.
DPPH activity (Antioxidant activity): The antioxidant activity of all extracts was checked by DPPH assay with the method given by Brand-Williams et al., (1995) . Ascorbic acid was used as the positive control. Color change of dye was observed and OD517 was recorded. Radical scavenging potential was calculated by %RSA= [(A0-AX)/A0] *100 (where A0 is the absorbance of negative control and AX is the absorbance of the sample).
α-glucosidase inhibitory activity: In vitro α-glucosidase inhibitory activity was carried out by the standard method presented by Phan et al., (2013). The activity was measured in the form of % inhibition by the formula (% Inhibition= [(OD0-ODS)/OD0] *100), where OD0 is the absorbance of negative control and ODs is the absorbance of the sample.
Antiviral activity
Inoculation of viruses in chicken embryonated eggs: In 9-10 days old chicken embryonated eggs, and selected viral strains were cultured through the chorio-allantoic route. The eggs were incubated at 37°C for 48-72 hrs. The allantoic fluid was collected and exposed to Haemagglutination (HA) to assess the titers of the virus.
Haemagglutination (HA) test: HA test was conducted by the method given by Killian, (2008). Red dots at the bottoms of the wells show negative results while uniform reddish color shows positive results.
Statistical analysis Data presented as mean ± SEM. One-way ANOVA was used for the analysis of data using Graph pad prism version 8.0.1. Dunnett’s multiple comparison test was used and values were compared with positive control where (P≤0.05)were considered significant (Ross et al., 2017).
RESULTS
Antibacterial activity: In terms of antibacterial activities MtOH extracts of all chosen Cholistani plants, exhibited strong antibacterial capability against selected bacterial strains. L. indicum was most effective against K. pneumoniae with a 14.5mm ZoI and MIC 390.62 µg/µl. It was least effective against P. aeruginosa (8mm ZoI) with notable effects against other strains as well. When E. granulata was evaluated for antibacterial properties, it was shown to be most efficient against E. coli with 16.5mm ZoI and MIC 1562.5 µg/µl. E. granulata was also shown to be particularly efficient against S. aureus and K. pneumoniae with 16 mm ZoI which was practically equivalent to positive control. The least inhibitory potential was seen against S. aureus MDR (7.5mmZoI) (7.5mmZoI). H. crispum exhibited maximum and minimum antibacterial potential against P. aeruginosa and P. aeruginosa MDR with 12 mm (MIC 1562.5 µg/µl) and 8.5mm ZoI. C. prostratus revealed generally strong antibacterial potential with 16mm ZoI (MIC 3125 µg/µl) against P. aeruginosa MDR and S. aureus (Table 1).
Table 1 Antibacterial activities of MtOH extracts of selected plants against drug sensitive and MRR strains.
Bacterial Strains
|
Plants
|
ZoI (mm)
|
MIC(µg/µl)
|
E. coli
|
L. indicum
|
11.5
|
|
E. granulate
|
16.5*
|
1562.5
|
H. crispum
|
10
|
3125
|
C. prostrates
|
9.5
|
3125
|
Ampicillin
|
17.5**
|
12500
|
|
Pooled SEM=0.82
|
|
|
K. pneumoniae
|
L. indicum
|
14.5
|
390.62
|
E. granulate
|
16*
|
|
H. crispum
|
10.5
|
|
C. prostrates
|
12.5
|
390.625
|
Ampicillin
|
16**
|
195.3
|
|
Pooled SEM=0.698
|
|
|
P. vulgaris
|
L. indicum
|
10
|
|
E. granulate
|
12*
|
|
H. crispum
|
10.5
|
390.62
|
C. prostrates
|
5
|
781.25
|
Ampicillin
|
13.5**
|
195.3
|
|
Pooled SEM=0.954
|
|
|
P. aeruginosa
|
L. indicum
|
8
|
|
E. granulate
|
14*
|
562.5
|
H. crispum
|
12
|
1562.5
|
C. prostrates
|
13
|
|
Ampicillin
|
15**
|
6250
|
|
Pooled SEM=0.30
|
|
|
S. aureus MDR
|
L. indicum
|
10
|
|
E. granulate
|
7.5
|
|
H. crispum
|
11
|
1562.5
|
C. prostrates
|
11.5*
|
3125
|
Moxifloxacin
|
16.5**
|
390.62
|
|
Pooled SEM=0.74
|
|
|
S. aureus
|
L. indicum
|
13.5
|
390.625
|
E. granulate
|
16*
|
3125
|
H. crispum
|
9
|
195.312
|
C. prostrates
|
13
|
|
Ampicillin
|
16.5**
|
6250
|
|
Pooled SEM= 0.698
|
|
|
P. aeruginosa MDR
|
L. indicum
|
11
|
781.25
|
E. granulate
|
8.5
|
|
H. crispum
|
8.5
|
|
C. prostrates
|
16*
|
3125
|
Moxifloxacin
|
10.5**
|
390.62
|
|
Pooled SEM=3.5
|
|
|
*indicates max ZoI
** indicate ZoI of positive control
Pooled SEM is calculated by adding log of sum of SEMs.
Antibiofilm activity: Antibiofilm potential of selected Cholistani plants was evaluated against drug sensitive and drug resistance strains of S. aureus and P. aeruginosa. In case of MDR strains E. granulata was most effective against S. aureus MDR with 84% inhibition whereas C. prostratus was highly effective against P. aeruginosa MDR with 79% suppression of biofilm development. When tested against drug sensitive strains, C. prostratus was the most efficient in preventing the growth of S. aureus biofilm, however H. crispum and L. indicum also showed significant suppression. C. prostratus also outperformed all other plant extracts in its ability to suppress the formation of P. aeruginosa’s biofilm by 90%, which was even more effective than the positive control (Table 2).
Table 2 Antibiofilm potential of MtOH extracts of different plants against selected bacterial strains. Data is presented as % inhibition± SEM.
Plant
|
S. aureus
|
S. aureus MDR
|
P. aeruginosa
|
P. aeruginosa MDR
|
% Inhibition± SEM
|
*Positive control
|
89.80±0.219
|
86.50±0.45
|
84.45±1.337
|
89.63±0.060
|
L. indicum
|
73.52±1.56
|
72.61±0.9
|
83.34±1.27
|
61.33±1.58
|
E. granulata
|
67.61±0.40
|
84.27±0.65
|
79.00±1.5
|
60.73±1.35
|
C. prostratus
|
81.60±0.21
|
83.79±0.25
|
90.09±0.039
|
79.64±0.51
|
H. crispum
|
74.49±1.73
|
76.922±1.39
|
74.55±1.2
|
72.18±0.418
|
*positive control= Ampicillin (Drug sensitive strains) and Moxifloxacin (MDR strains)
Antioxidant Activity: The antioxidant potential of selected plants was observed by DPPH assay and %RSA was calculated for each plant. It was observed that E. granulata possessed the highest scavenging potential i.e., 82%. Other plants, in the following order: C. prostratus > L. indicum > H. crispum, showed the ability to scavenge radicals: 57%, 53%, and 51.34%RSA, respectively. The ascorbic acid was used as positive control (Fig 1).
α- glucosidase Inhibitory assay: According to the antidiabetic potential of MtOH extracts of selected plants was checked by α- glucosidase inhibitory assay, C. prostratus and E. granulata were found to be most effective with 82% and 81% inhibitions respectively. Whereas, L. indicum (73%) and H. crispum (61%) showed considerable inhibition potential values. Acarbose was used as positive control and exhibited 95% inhibition against α- glucosidase (Fig 2).
Antiviral activity: MtOH extracts of selected plants were subjected to an HA (Hemagglutination) test for antiviral potential evaluation against AIVH9N2. E. granulata and C. prostratus were equally effective with 0 titers (<log2). In the case of other plants, H. crispum was more effective than L. indicum with 4 (8 logs) and 8 (7 logs) titers respectively (Table 3).
Fig. 1 %Radical scavenging activity of Methanolic extracts of selected plants.
Values are presented as %RSA± standard error. ****(P<0.0001) and **(P=0.0137).
Fig 2 α- glucosidase Inhibitory potential of Methanolic extracts of selected Cholistani plants.
Values are presented as %Inhibition± SEM. ****(P<0.001), ***(P<0.002) and **(P<0.0011). Table 3 Antiviral activity of MtOH extracts of selected plants against AIVH9N2.
Plants
|
HA Titer
|
Log reduction
|
IC50 µg
|
L. indicum
|
8
|
7
|
500
|
E. granulate
|
0
|
<log2
|
125
|
C. prostrates
|
0
|
<log2
|
125
|
H. crispum
|
4
|
8
|
250
|
Virus control
|
1024
|
0
|
|
Amantadine
|
0
|
<log2
|
125
|
*Log reduction= [log2 (control=10)-log2 Sample]
DISCUSSION
The desert offers a range of plants that are helpful to fight against numerous disorders (Malik et al., 2015). Antibiotic resistance among microbial strains is fostered by the widespread use of antibiotics in clinical treatment, agriculture, and veterinary care (Hussain et al., 2015). This has led to the investigation of novel antimicrobial agents, mainly among plant extracts, in order to produce new medications that overcome the disadvantages of antibiotics (Bereksi et al., 2018). In the present work, the antibacterial potential of MtOH extracts of four Cholistani plants E. granulata, C. prostratus, L. indicum, and H. crispum was examined. E. granulata dominated antibacterial activity but other plants also shown great potential, suggesting that these plants may become a suitable alternative to antibiotics (Ihtesham et al., 2019; Indrianingsih et al., 2021).
Due to various therapeutic issues connected with conventional antibiotics, there has been a rise in interest in utilizing plant extracts for the treatment of biofilm-related disorders. Herbal plants are excellent source of alternative remedies for a number of ailments (Wijesundara and Rupasinghe, 2019). In the current investigation C. prostratus displayed the strongest antibiofilm potential, whereas L. indicum, E. granulata, and H. crispum showed potential in varied order. (Chouhan et al., 2023) also reported that C. prostratus possessed great antibacterial potential against selected bacterial strain. The efficiency of these Cholistani plants against MDR strains implies that these plants are the significant source of antimicrobial agents against biofilm-forming resistant pathogens (Song et al., 2018).
Antioxidants act as a key defense against the substantial oxidative damage caused by the reactive oxygen species in the body (Venkatachalam et al., 2012). Extensive studies are being performed to examine the antioxidant potential of medicinal plants. According to the present research, E. granulata exhibited the greatest antioxidant potential of all other selected plants investigated, with 82% RSA. Al-Robai et al., (2023) conducted DPPH assay of E. granulata and reported that is a potent source of antioxidant compounds. C. prostratus, L. indicum, and H. crispum have also exhibited strong antioxidant potential.
Diabetes is a prominent cause of death and morbidity globally. Antidiabetic medicines are more effective in the early stage of diabetes and are often used in combination with other drugs. Therefore, individuals are inclined toward the usage of herbal medications for diabetes (Elbashir et al., 2018). In the present investigation, C. prostratus and E. granulata displayed maximum inhibitory potential but L. indicum and H. crispum were also regarded as effective (Arshad et al., 2021). A similar study was conducted where hydro-alcoholic extract of Euphorbia Hirta exhibited maximum inhibition of alpha- glucosidase enzyme (Sheliya et al., 2016).
Due to unsatisfactory and restrictive therapy alternatives, there is an ever-increasing demand for antiviral drugs. Consequently, medicinal plants, with a wide range of phytochemicals, have substantial potential to solve this problem (Zitterl-Eglseer and Marschik, 2020). All of the selected Cholistani plants tested positive for antiviral activity against AIVH9N2, notably E. granulata and C. prostratus, highlighting the significance of these plants as natural sources of antiviral drugs.
Conclusion: Cholistan plants Limeum indicum, Heliotropium crispum, Convolvulus prostratus and Euphorbia granulata are a rich source of antibacterial, antibiofilm, antioxidant, antidiabetic, and antiviral compounds. Additionally, these medicinal plants have proved their worth as a low-cost and feasible alternative drug source to present medicines.
Author’s Contribution: Mirza Imran Shahzad, Fatima Sadiq and Hadeeqa Habib conceived and designed the study. Muhammad Abdullah identified the selected plants. Kiran Fatima and Muneeza Mustafa executed the antibacterial activity. Haleema Saeed and Maryam Shafiq evaluated the antioxidant potential of selected Cholistani plants. α-glucosidase assay was performed by Saba Ajmal and Tamawul Noor. Antiviral activity of the selected plants was performed by Irfan Saeed and Sajid Hussain. Hina Ashraf and Fatima Sadiq checked the antibiofilm potential of selected Cholistani plants. All authors interpreted the data, critically revised the manuscript for important intellectual contents and approved the final version.
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