|Year : 2014 | Volume
| Issue : 2 | Page : 95-103
Pharmacological evaluation of calendula officinalis L. on bronchial asthma in various experimental animals
Rakesh Sagar1, Himanshu Bhusan Sahoo2, Biswakanth Kar3, Nikunja Kishor Mishra4, Rajaram Mohapatra5, Sarada Prasad Sarangi6
1 Department of Pharmaceutical Sciences, Dr. H.S. Gour Vishwavidyalaya, Sagar, Madhya Pradesh, India
2 Department of Pharmacology and Experimental Biology, Vedica College of Pharmacy, Ram Krishna Dharmarth Foundation University, Bhopal, Madhya Pradesh, India
3 Department of Pharmaceutical Technology, Jadavpur University, Kolkata, West Bengal, India
4 School of Pharmaceutical Education and Research, Berhampur University, Berhampur, Odisha, India
5 School of Pharmaceutical Science, Siksha O Anusandhan University, Bhubaneswar, Odisha, India
6 Department of Pharmaceutical Chemistry, Jayamukhi Institute of Pharmaceutical Science, Warangal, Andhra Pradesh, India
|Date of Submission||29-Nov-2013|
|Date of Acceptance||05-Jan-2014|
|Date of Web Publication||29-Mar-2014|
Himanshu Bhusan Sahoo
Department of Pharmacology and Experimental Biology, Vedica College of Pharmacy, Ram Krishna Dharmarth Foundation University, Bhopal 462 033, Madhyapradesh
Source of Support: Vedica College of Pharmacy, RKDF University,
Bhopal - 462 033, Madhya Pradesh, India,, Conflict of Interest: None
| Abstract|| |
Background: Calendula officinalis Linn. (Asteraceae) is an aromatic herb growing in the forests of India, China, Central Europe, and some tropical areas. The study was designed to investigate the scientific basis for the traditional claim of C. Officinalis on asthma. In the present study, methanol extract of whole plants was evaluated for preliminary phytochemical screening and antiasthmatic activity on different animals. Subjects and Methods: The asthmatic activity was evaluated by histamine- or acetylcholine-induced bronchospasm in guinea pigs, compound 48/80-induced mast cell degranulation in wistar rats, histamine-induced constriction on isolated guinea pig trachea, and ovalbumin-induced sensitization in mice at different dose levels of C. Officinalis. The preconvulsion dyspnoea time at 0 th and 7 th days at the dose of 250 and 500 mg/kg in guinea pig, the percentage of granulated and degranulated mast cell of at the dose of 600, 800, 1000 μg/mL in rats, muscular contraction at the dose of 600, 800, and 1000 μg/mL on isolated guinea pig trachea and the inflammatory cell count, that is, eosinophils, neutrophills, lymphocytes, macrophages, interleukin (IL)-4, IL-5, and immunoglobulin-E (IgE) from bronchoalveolar lavage fluid at the dose levels of 50, 100, and 250 mg/kg in mice were evaluated and compared with respective control groups. Results: Phytochemical studies revealed the presence of flavonoids, steroids, saponin, terpenoids, lignins, and phenolic compounds in the extract. In addition, the treatment of methanolic extract of C. officinalis (MECO) significantly (P < 0.001) decreased the bronchospasm induced by histamine or acetylcholine in guinea pigs, degranulation mast cell in rats, histamine-induced constriction on isolated guinea pig trachea, and the level of inflammatory cells as compared with inducer groups. The antiasthmatic activity was potentiated in all the doses in dose-dependent manner. Conclusion: The present study concludes that the antiasthmatic activity may be due to the presence of above phytoconstituents by antihistaminic, anticholinergic, antispasmodic, and mast cell stabilizing property.
Keywords: Asthma, bronchospasm, C. Officinalis, mast cell degranulation, ovalbumin
|How to cite this article:|
Sagar R, Sahoo HB, Kar B, Mishra NK, Mohapatra R, Sarangi SP. Pharmacological evaluation of calendula officinalis L. on bronchial asthma in various experimental animals. Int J Nutr Pharmacol Neurol Dis 2014;4:95-103
|How to cite this URL:|
Sagar R, Sahoo HB, Kar B, Mishra NK, Mohapatra R, Sarangi SP. Pharmacological evaluation of calendula officinalis L. on bronchial asthma in various experimental animals. Int J Nutr Pharmacol Neurol Dis [serial online] 2014 [cited 2019 Nov 13];4:95-103. Available from: http://www.ijnpnd.com/text.asp?2014/4/2/95/129595
| Introduction|| |
Asthma is one of the most common chronic inflammatory disorders of the airways; characterized by inflammation or obstruction in throat, bronchial hyperresponsiveness, and mucosal hypersecretions.  The prevalence of asthma has significantly increased in recent decades, that is, nearly 7%-10% of the world population were affected in each year. It can be triggered by various factors like allergens, drugs, respiratory infection, dust, cold air, exercise, emotions, occupational stimuli, chemicals, histamine, and also hereditary. , These trigger factors accelerates the activation of immunoglobulin-E (IgE)-mediated mast cell, release of interleukins (IL-4 and IL-5) and other inflammatory factors, including eosinophils, neutrophills, B cells, cytokines, and chemokines. , Therefore, the disease statistics clearly necessitates the drugs targeting toward cytokine inhibitors, neutralizing antibodies directed IgE, histamine and leukotriene blockers, and so on for the management of asthma. , Despite the availability of a wide range of antiasthmatic drugs, the relief offered by them is mainly symptomatic and short lived with more or less side effects. Assessing the current status of health care system, there has been an alarming increase in number of diseases or disorders caused by synthetic drugs prompting a switch over to traditional herbal medicine. , In this regard, natural compounds isolated from medicinal plants having greater antioxidant, anti-inflammatory and immunomodulatory activity was recognized as a gold candidate for asthma. , So, an ideal approach in development of new drug toward safe and effective remedies is only from herbal sources to treat bronchial asthma.
Calendula officinalis Linn. popularly known as Pot Marigold, (Asteraceae) is an annual or biennial aromatic herb distributed in the moist deciduous forests of India, China, Central Europe, and some tropical areas.  The whole plant grows a height up to 30-60 cm with soft, glandular, and attractive leaves, with bright yellow or orange flowers. The stem is slightly fuzzy and the leaves are long spindly taproot with bitter taste. All parts are highly scented and, therefore, attractive to been and hover slies.  It is an important medicinal plant, has been widely used for treatment of many diseases in homeopathic or ayurvedic system of medicine.  In Chinese medicinal system, it is a gentle remedy that helps to prevent the development of bacterial and fungal sinus infections. Also used for treatment of scorpion bites and prevention for gangrene by the Roman traditional practitioners.  It has been reported to possess many pharmacological activities, which include antioxidant,  anti-inflammatory,  antibacterial,  antifungal,  antiviral,  aphrodisiac,  antigenotoxic,  antihypertensive  antispasmodic,  hypoglycaemic  and cytotoxic as well as tumor-protective potential.  It is used as analgesic, anthelmintic, antiemetic, antipyretic, antiseptic, expectorant, astringent, bitter, cardiotonic, carminative, cholagogue, dermagenic, diaphoretic, diuretic, hemostatic, immunostimulant, lymphatic cleanser, uterotonic, and as vasodilator. Generally, it is used as a best antiseptic due to its natural content of iodine, so applied externally on skin inflammations, open wounds, burns, or cuts. , Internally it is used for mucous membrane inflammations, peptic and duodenal ulcers, spasms of the gastrointestinal tract, splenic and hepatic inflammations, duodenal and intestinal mucosa, and dysmenorrhea especially in nervous or anaemic women. It is also used as a mouthwash after tooth extractions. Calendula oil is an important ingredient of many cosmetic preparations, especially for cosmetic care of sensitive and dry skin. , The plant is rich in many pharmaceutical active ingredients like flavonoids, carotenoids, glycosides, alkaloids, triterpinoids, coumarines, volatile oils, sterols, polysaccharides, and amino acids. It contains calanduline, lupeol, quercetin, protocatechuic acid etc. , On the basis of above-mentioned scientific evidences, the present study was undertaken to investigate the effect of the whole plant extract of Calendula officinalis L. for its antiasthmatic activity on different animal models.
| Subjects and Methods|| |
Chemicals and reagents
Histamine dihydrochloride, acetylcholine chloride, ovalbumin, ketotifen, compound 48/80 were purchased from Sigma-Aldrich Chemical Co., USA. Egg albumin, aluminium hydroxide, and other chemicals were purchased from Himedia Laboratories Pvt. Ltd., India. All the other chemicals were of analytical grade.
Plant material and preparation of the extract
The whole plant of Calendula officinalis Linn. was collected from local area of Bhopal, Madhya Pradesh, India. The species was identified and authenticated at the Department of Botany, Dr. H. S. Gour Central University, Sagar (M.P), where a plant specimen was deposited. The whole plants were allowed to air dry, powdered and weighed (1 kg). After defatting with petroleum ether, the drug was subjected to continuous hot extraction in soxhlet apparatus using methanol as solvent for 48 h. After complete extraction, the extract was filtered and evaporated under reduced pressure to obtain dried methanolic extract (19.28% w/w). For antiasthmatic evaluation, extract was dissolved in distilled water prior to its use.
Preliminary phytochemical tests were performed on methanolic extract of C. Officinalis (MECO) for the presence of various phytoconstituents. 
Wistar rats (175-200 g), Balb/c mice (25-30 g) and guinea pigs (400−600 g) of either sex housed in standard conditions of temperature (22 ± 2°C), relative humidity (55 ± 5%), and light (12 h light/dark cycles) were used. They were fed with standard pellet diet and water ad libitum. The experimental protocol was approved by Institutional Animal Ethical Committee as per the guidance of CPCSEA, Ministry of Social Justice and Empowerment, Government of India.
Acute toxicity testing
The animals were fasted overnight prior to the experiment. Different-graded doses of methanolic extract were administered orally to animal groups and were observed continuously for 24 h for any gross behavioural changes, followed any mortality as per the OECD Guideline 425. [ 33 ]
Histamine-and acetylcholine-induced bronchospasm in guinea pigs
Guinea pigs of either sex were divided into two groups, each group containing six animals and exposed to 0.1% w/v of histamine dihydrochloride aerosol in histamine chamber. The progressive dyspnoea was observed in animals when exposed to histamine aerosol. The end point, preconvulsion dyspnea (PCD) was determined from the time of aerosol exposure to the onset of dyspnea leading to the appearance of convulsion. As soon as PCD commenced, the animals were removed from chamber and placed in fresh air. PCD of this time was taken as day 0 value. Both groups of guinea pigs were given methanolic extract of C. officinalis at the dose of 250 mg/kg, and 500 mg/kg, p.o. respectively, once a day for 7 days. On the 7 th day, 2 h after the last dose, the time for the onset of PCD was recorded as on day 0. Same procedure was followed in another set of animals (n = 6) for acetylcholine induce bronchospasm study except using 0.5% acetylcholine chloride in place of histamine dihydrochloride.  The percentage increased in time of PCD was calculated using following formula.
Percentage increased in time of PCD = (1-T 1 /T 2 ) × 100
Where: T 1 = time for PCD onset on day 0, T 2 = time for PCD onset on day 7.
Mast cell degranulation studies
Albino rats of either sex were divided into six groups; each group carries six animals and sacrificed by cervical dislocation. The animals were immediately injected with 15 mL of prewarmed (37°C) buffered salt solution (NaCl 137 mM; KCl 2.7 mM; MgCl 2 ; 1 mM; CaCl 2 0.5 mM; NaH 2 PO 4 0.4 mM; glucose 5.6 mM; HEPES 10 mM) into the peritoneal cavity, and massaged gently in this region for 90 s, to facilitate cell recovery. A midline incision was made and the peritoneum was exposed. The pale fluid was aspirated using a blunted plastic Pasteur pipette, and collected in a plastic centrifuge tube. The fluid was then centrifuged at 1000 rpm for 5 min, and the supernatant was discarded to reveal a pale cell pellet. The cell pellets were resuspended in fresh buffer and recentrifuged.
The peritoneal cell suspension divided in six parts viz. -ve control, +ve control, reference standard (ketotifen 10 μg/mL) and methanolic extract of C. officinalis at different concentration, that is, 600, 800, 1000 μg/mL, each containing 0.1 mL of cell suspension and incubated at constant temperature 37°C in water bath for 15 min. Then, 0.1 mL of compound 48/80 was added in all samples except in -ve control group and suspensions were further incubated for 10 min at 37°C. The cells were then stained with 10% of toluidine blue solution and observed under the higher magnification by microscope. The percent granulated and degranulated mast cells were counted in each group. 
Guinea pig tracheal chain preparation
Guinea pigs of either sex (200-500 g) were divided into four groups. Each group contains six animals and were allowed to starve overnight and free access to water. The animals were killed by a blow on the head and exsanguinated. The isolated trachea was mounted in a 30 mL organ bath containing tyrode solution, maintained at 37 ± 1°C and gassed with air. The tissue was equilibrated for 45 min during which the bath solution was replaced every 10 min. At the end of the equilibration period, histamine (0.5 μg/mL)-induced contraction as well as effect of extract (up to 1000 μg/mL) was recorded. A drug tissue contact time of 1 min was maintained. The percent response of each groups were calculated from the height of the peaks obtained. 
Ovalbimin-sensitization from bronchoalveolar lavage fluid in mice
The mice were divided into five groups, each group containing six mice. The control group received only phosphate buffered saline (PBS) (vehicle); another control group treated with AL (OH) 3; Positive control group was immunized by subcutaneous injection of a suspension containing 100 μg of ovalbumin and 1 mg AL (OH) 3 in 200 μL of PBS on days 1 and 7; and the treatment groups were sensitized by ovalbumin and then intraperitoneally treated with 50, 100, and 250 mg/kg of extract on days 8-14 separately. The mice were sensitized by subcutaneous administration of 100 μg of ovalbumin emulsion (using aluminium hydroxide and PBS). The efficiency of sensitization was assessed by measurement of ova-specific IgE levels from mice. For evaluation of ova-specific IgE levels, serum samples were collected of the mice on 14 th day. The absorbance was measured at 450 nm by microplate enzyme-linked immunosorbent assay (ELISA) reader. On day 15, lung lavaging was performed for preparation of BALF. The thorax cavity of mouse was opened and then sheered the omohyoid and stylohyoid muscles, then for prevention of lavage reflux. A needle or a fine polyethylene tube was fixed in trachea and 1 mL of PBS was injected to the fixed tube via insulin syringe and then it was aspirated (three time) until 2 mL of BALF was taken. The suspension of BALF was centrifuged and the supernatant collected and stored at − 70°C. Inflammatory cell numbers including eosinophil, lymphocyte, neutrophil, macrophage, and total cells were determined by direct microscopic counting with a hemocytometer after exclusion of dead cells by trypan blue staining. In order to determine the levels of IL-4, IL-5 in BALF was measured by ELISA kits (Allied Biotechnology India Pvt. Ltd., Mumbai, India). ,
The results of various studies were expressed as mean ± standard error of the mean and analyzed statistically using one-way analysis of variance followed by student's t-test to find out the level of significance. Data were considered statistically significant at minimum level of P < 0.001.
| Results|| |
Preliminary qualitative phytochemical screening of MECO showed the presence of flavonoids, phenols, steroids, tannins, terpenoids, saponins, lignins, amino acids, and carbohydrates.
Acute toxicity study
No mortality and the sign of toxicity were observed at the dose of 6000 mg/kg in case of albino rats.
Effect of MECO on histamine and acetylcholine aerosol-induced bronchospasm in guinea pigs
MECO was significantly and dose dependently increased the time of PCD following exposure to histamine (P < 0.001) and acetylcholine (P < 0.001) aerosol induced bronchospasm in guinea pigs [Table 1]. The percentage of increase in PCD in histamine induced bronchospasm at the dose of 250 mg/kg and 500 mg/kg body weight was found 69.19% and 78.98% respectively, whereas in case of acetylcholine induced bronchospasm at the same dose level was 57.13% and 63.13%, respectively. So, the percentage of increase of PCD was more against histamine induced bronchospasm as compared with acetylcholine by the administration of C. Officinalis.
|Table 1: Effect of methanolic extract of Calendula officinalis on histamine-and acetylcholine-induced bronchospasm in guinea pigs |
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Effect of MECO on compound 48/80 induced mast cell degranulation in rats
The percentage of mast cell degranulation was observed [Figure 1] in groups of -ve control (9.49 ± 1.623), +ve control (75.26 ± 1.426), ketotifen as standard (23.87 ± 2.178), MECO I-600 μg/mL (58.22 ± 2.422), MECO II-800 μg/mL (43.66 ± 2.899) and MECO III-1000 μg/mL (30.88 ± 2.826). The treated groups of MECO as well as the standard group were observed significant (P < 0.001 and P < 0.01) inhibition of mast cell degranulation from rat peritoneal cell. The treated groups of MECO also able to dose-dependent mast cell protection against compound 48/80 as compared with base line value of positive control.
|Figure 1: Effect of methanolic extract of Calendula officinalis on compound 48/80-induced mast cell degranulation in albino rats. Values were expressed as mean ± standard error of the mean, where n = 6 in each group, * P < 0.001, # P < 0.01when compared with positive control group|
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Effect of MECO on Guinea pig tracheal chain
In isolated guinea pig tracheal studies, it significantly (P < 0.001) inhibits the contraction of tracheal muscles induced by histamine as compared to control group in dose-dependent manner [Figure 2]. In MECO-treated groups, the percentage of inhibition was found to be 31.64%, 43.71%, and 67% as compared with histamine-treated group.
|Figure 2: Effect of methanolic extract of Calendula officinalis on percentage of inhibition in histamine-induced constriction on isolated guinea pig trachea|
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Effect of MECO on inflammatory cells from BALF in ovalbumin sensitized in mice
After challenging with ovalbumin, the levels of inflammatory cells, that is, eosinophils, neutrophills, macrophages, lymphocytes, and total cells were significantly increased (P < 0.01) in ovalbumin treated group as compared with control group. But, sensitization of ovalbumin along with MECO treatment, the levels of inflammatory cells were significantly (P < 0.01) and dose dependently decreased as compared with ovalbumin treated group [Figure 3].
|Figure 3: Effect of methanolic extract of Calendula officinalis on inflammatory cells in the bronchoalveolar lavage fluid of mice. Values were expressed as mean ± standard error of the mean, where n = 8 in each group, *P < 0.01 Compared with control (vehicle only), #P < 0.01 compared with ovalbumin-treated groups|
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Effect of MECO on serum IgE and Interleukins (IL-4 and IL-5) level in ovalbumin Sensitized in mice
In the present study shown that the levels of IL-4 and IL-5 in BALF were significantly increased in ovalbumin treated group as compared with control group (P < 0.01), whereas these levels were protected in dose-dependent manner in MECO-treated groups with coadministration of ovalbumin [Figure 4]. Similarly, the serum IgE level was also raised due to sensitization of ovalbumin in ovalbumin treated group. But, ovalbumin-specific IgE levels (pg/mL) were significantly as well as dose dependently reduced in MECO treated groups [Figure 5].
|Figure 4: Effect of methanolic extract of Calendula officinalis on the levels of interleukin (IL)-4 and IL-5 in bronchoalveolar lavage fluid. Values were expressed as mean ± standard error of the mean, where n = 8 in each group, *P < 0.01 compared with control (vehicle only), #P < 0.01 compared with ovalbumin-treated group|
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|Figure 5: Effect of methanolic extract of Calendula officinalis on ovalbumin-specific immunoglobulin E levels in serum. Values were expressed as mean ± standard error of the mean, where n = 8 in each group, *P < 0.01 compared with control (vehicle only), #P < 0.01 compared with ovalbumin-treated groups|
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| Discussion|| |
MECO was evaluated for antiasthmatic activity using different screening animal models as asthma involves various types of mediators. Bronchial asthma is commonly characterized by increased airway reactivity to different spasmogens. An initial attack of asthma was triggered by the release of inflammatory mediators like histamine, acetylcholine, leukotrienes, prostaglandins, or specific exposure of allergens, which reflected the signals of acute bronchoconstriction. , The close resemblance of pulmonary responses to histamine challenge in both guinea pigs and humans, as well as the anaphylactic sensitization made this species the model of choice. In the present study, aerosolized histamine and acetylcholine were used to cause immediate bronchoconstriction in the form of PCD in guinea pigs. Bronchodilating effect of MECO was evaluated by observing its effects on the time of PCD. The study found that the time of occurrence of PCD was significantly increased that suggests bronchodilating activity of C. officinalis against spasmogens.
Mast cell degranulation is an important factor for initiation of immediate responses following exposure to allergens. Once binding of allergen to cell-bound IgE occurs, the mast cell releases the inflammatory mediators such as histamine, eosinophils, and neutrophils chemotactic factors, leukotrienes, prostaglandins, platelet-activating factor, and so on; which are responsible development of airway inflammation and bronchoconstriction.  Here, an attempt was made to find out whether MECO has any effect on the rate of disruption of mast cells following exposure to compound 48/80, an agent which causes histamine release. In this study, MECO offered significant protection against compound 48/80-induced mast cell degranulation by stabilizing it, which is responsible for the decreasing airway inflammation by preventing release of various inflammatory mediators. The relaxant effects of on tracheal chains of guinea pigs might be produced by different mechanisms including stimulation of β-adrenergic receptors, inhibition of histamine (H1) receptors, or an anticholinergic property of this plant.  The relaxant effects of all concentrations of MECO obtained were significantly lower than control group. These findings suggest probable β-adrenergic stimulatory, muscarinic or histamine (H 1 ) blocking properties of the plant extract.
The elevated number of the inflammatory cells like eosinophils, neutrophills, lymphocytes, monocytes in blood and biopsies of lung tissue, bronchial alveolar lavage fluid or sputum reflects the sign of asthma. The results of this study showed that the treatment of MECO (50, 100, and 250 mg/kg) with ovalbumin sensitized mice significantly reduced the levels of eosinophils, neutrophills, macrophages, lymphocytes, and total inflammatory cells in the BALF as compared with ovalbumin sensitized mice. Lymphocytes play an important role in the initiation and progression of allergic asthma by releasing of IL-4 and IL-5 cytokines.  These lymphocytes-induced airway inflammatory cells infiltration, eosinophils activation, IgE production, and mucus secretion, which resulted as bronchial hyperactivity.  On the contrary, serum IgE level is associated with Th2 response.  IL-4 cytokine is important to the induction of isotype class switching which is required for B cells to express IgE.  IL-5 cytokine is pivotal for growth, differentiation, recruitment, and survival of eosinophils.  In the present study, MECO treatment in ovalbumin-sensitized mice significantly decreased the levels of IL-4 and IL-5 cytokines in the BALF comparing with ovalbumin sensitized mice. The decrease of IL-5 in the BALF of the mice that had been treated with all extracts of C. officinalis and ovalbumin-treated groups may at least in part be responsible for the reduced recruitment of eosinophils. These results show that the protective effect of MECO by preventing the infiltration of inflammatory cells, by decreasing the release of preformed inflammatory mediators, which can prevent the direct damage to airway and its hyperresponsiveness.
Phytochemical screening of MECO showed the presence of flavonoids, steroids, lignins, saponins, terpenoid derivatives, and so on. Several flavonoids are known to possess various biological activities, including smooth muscle relaxant, bronchodilator, antibacterial, spasmolytic, and anti-inflammatory effects, ,, whereas lignins are responsible for antibacterial, antioxidant and anticancer, spasmolytic and anti-inflammatory effects.  Saponins, steroids, and terpenoids were responsible for bronchospasmolytic action by relaxing tracheobronchial tree of lungs. , The antiasthmatic activity of MECO may be due to presence of the above constituents.
In conclusion, the results of this study suggest that the MECO possesses anticholinergic, antihistaminic, mast cell protection against degranulation and anaphylactic activity in different animal models. These pharmacological activities collectively constitute significant prevention against asthma. However, further studies are needed to establish molecular mechanism and to isolate or characterize the bioactive compounds, which are mainly responsible for the antiasthmatic action.
| Acknowledgment|| |
We are grateful to the Director, Vedica college of B. Pharmacy, Bhopal, MP; for providing the lab facilities and guidance during the course of this study.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]