Laxative potential of the ethanolic leaf extract of Aloe vera (L.) Burm. f. in Wistar rats with loperamide-induced constipation

A.O.T Ashafa, TO Sunmonu, AA Abass, AA Ogbe

Journal of Natural Pharmaceuticals 2011 2(3):158-162

Background: Constipation is a highly prevalent and often chronic functional gastrointestinal disorder affecting humans irrespective of race and color exposing victims to colorectal cancer. The present study evaluated the efficacy of the ethanolic leaf extract of Aloe vera against loperamide-induced constipation in rats. Materials and Methods: Rats were constipation induced by the oral administration of loperamide (3 mg/kg body weight) while the control animals received normal saline. Constipated rats were treated with 50, 100, and 200 mg/kg body weight/day of the ethanolic leaf extract for 7 days during which the feeding characteristics, body weight, fecal properties, and gastrointestinal transit ratio were monitored. Results: Treatment of constipated rats with the extract at 50, 100, and 200 mg/kg body weight for 7 days improved intestinal motility, increased fecal volume, and normalized body weight in the constipated rats. These are indications of the laxative property of the herb with the 200 mg/kg body weight of the extract showing the best efficacy. Conclusion: The effect of the extract compares favorably well with Gaviscon, a standard laxative drug. These findings have therefore lent scientific support to the use of the herb as a laxative agent in Nigerian folkloric medicine.

Effect of oral administration of ethanolic root extract of Tinospora cordifolia on aflatoxin B 1 -induced toxicity in swiss albino mice

Veena Sharma, Rekha Gupta, Shatruhan Sharma

Journal of Natural Pharmaceuticals 2011 2(3):125-132

Background: Aflatoxins are potent hepatotoxic and hepatocarcinogenic agents. This hepatotoxicity is thought to be mediated by their ability to generate reactive oxygen species and cause peroxidative damage. Considering the antioxidant properties of Tinospora cordifolia, this study was undertaken to evaluate the therapeutic efficacy of Tinospora cordifolia root extract in terms of altered biochemical, hematological, serological and histopathological parameters. Methods: Sixty male swiss albino mice were taken for post exposure therapy. Results: Aflatoxin exposure elicited a significant escalation in the level of thiobarbituric acid reactive substances and depletion in reduced glutathione, protein, ascorbic acid and antioxidant enzymes, namely, superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and glutathione S-transferase activities. Chronic aflatoxin ingestion showed a significant decline in total erythrocyte count, lymphocyte count, hemoglobin, hematocrit values while total leukocyte count, platelet count and neutrophil count significantly increased in the aflatoxin-treated group. The activities of aspartate transaminase, alanine transaminase and alkaline phosphate augmented significantly in the serum of aflatoxin-exposed mice suggesting hepatic damage. Aflatoxin administration decreased the content of high density lipoprotein while increased the content of cholesterol, triglyceride, low density lipoprotein and very low density lipoprotein significantly. Pathological examination of the liver tissue also supported the biochemical findings. However, post-exposure administration of Tinospora cordifolia root extract to the aflatoxin-treated group attenuated the deranged parameters to some extent. Conclusion: This study indicated that Tinospora cordifolia can be a protective regimen for aflatoxin toxicity.

Comparative toxicity assessment of three Tephrosia species on Artemia salina and animal cell lines

Sandhya Subhadra, Venkata Ramana Kanacharalapalli, Vinod Kombath Ravindran, Sai Kumar Parre, Sunitha Chintala, Rajeswar Thatipally

Journal of Natural Pharmaceuticals 2011 2(3):143-148

Background: Tephrosia calophylla, Tephrosia maxima and Tephrosia purpurea are generally used in the treatment of various ailments including cancer by the folklore of Indian subcontinent. Materials and Methods: The powdered root of three plants were separately extracted with chloroform and then subjected to preliminary chemical screening. The total phenolic and flavonoid content were estimated by using gallic acid and quercetin as standard. Brine shrimp hatchability and lethality tests were done by hatching the cysts of Artemia salina in brine solution. As these parameters can be associated to tumor cells, the investigation was extrapolated to animal cell lines, Daltons Lymphoma Ascites (DLA) and Erlisch Ascites Carcinoma (EAC). Trypan blue exclusion method was adopted for this screening. In all experiments dose selected were between the ranges of 10 and 200 &#956;g/mL. Results: Preliminary chemical screening revealed the presence of carbohydrates, proteins, alkaloids, tannins and flavonoids for T. calophylla and T. maxima; proteins, alkaloids, tannins and flavonoids for T. purpurea. The total phenolic and flavonoid content was found to be highest in the case of T. calophylla followed by T. maxima and then by T. purpurea. It was observed that in all the cytotoxicity tests performed T. calophylla was proved to be most potent followed by T. maxima and then by T. purpurea. The results thus obtained were found to correlate with the values obtained for total phenolic and flavonoid content. Conclusion: The present investigation revealed that all three species of Tephrosia to be potent cytotoxic agents. Increasing order of cytotoxicity can be denoted as T.purpurea < T.maxima < T.calophylla. Further studies have to be performed to identify the chemical entity responsible for efficient cytotoxicity which may help in the development of suitable lead compounds.

Flavonoids inhibited NADPH consumption and ecdysis processes in Oncopeltus fasciatus

Juliana Oliveira Abreu Narciso, Marco Antonio Soares de Souza, Mario Geraldo de Carvalho, Mário Sergio da Rocha Gomes, Marcelo Genestra, Marise Maleck de Oliveira Cabral

Journal of Natural Pharmaceuticals 2011 2(3):133-137

Background: Piptadenia rigida is a source of flavonoids such as isoliquiritigenin ( 1 ), 7,3',4'-trihydroxyflavone ( 2 ) and 7,8,3',4'-tetrahydroxyflavanone ( 3 ). Flavonoids influence on the feeding behavior of insects besides the inhibition of the insect larvae growth. Nitric oxide (NO) seems to be conserved in invertebrate innate immunity and the NO synthase (NOS) activity has been implicated in insect immunity. Therefore, the NOS expression can be evaluated to determine the inhibition of NADPH consumption. Material and Methods: three natural flavonoids, isolated from P. rigida whose structures were determined by 1 H and 13 C NMR spectral data analysis, were evaluated on the Oncopeltus fasciatus control by molting processes and NADPH consumption in the insect intestine following mortality. Results: The flavonoids treatment on O. fasciatus showed 50% mortality and 50% ecdysis ( 1 ), 30% mortality and 43% ecdysis ( 2 ), and topical treatment with th 3 resulted in 43% ecdysis but did not show high toxicity at 100&#956;g/nymph. Intestine homogenates obtained from insects treated with flavonoids that were incubated with NADPH substrate showed percentage inhibitions of 72%, 78% and 80%, for the treatments 3, 1 and 2, respectively. Conclusion: The flavanone ( 3 ) was the most effective and least toxic to the insect, followed by 2 and then 1 .

HPLC analysis of protoberberine alkaloids in Phellodendron lavallei D. introduced to Western Georgia

Mariam Meskheli, Valentina Vachnadze, Dali Berashvili, Aliosha Bakuridze

Journal of Natural Pharmaceuticals 2011 2(3):154-157

Background : Phellodendron lavallei D. is one of the oldest medicinal plants and is of current interest as a natural medicine. The aim of our study was to determine the features and amount of the content of berberine, palmatine, jatrorrhizine, and magnoflorine in the overall preparation enriched by pharmacologically active alkaloids, obtained from the bark of P. lavallei, using HPLC. Materials and Methods: The chromatographic separation was carried out using a Zorbax SB-C18 column (250 &#215; 4.6 mm, 5 &#956;m; Waters), mobile phase MeOH 20 mL/mol and KH 2 PO 48:52 (pH 2.6). The UV detection was performed at 235 nm. Results: A good linear behavior over the investigated concentration range was observed with the value r2 > 0.9999. The amount of palmatine, jatrorrhizine, magnoflorine, and berberine in the extract was determined and the following results were achieved: berberine 7.6%, palmatine 0.77%, jatrorrhizine 0.9%, and magnoflorine 57%. Conclusion: Study results show that this method of HPLC might be used to determine alkaloids such as berberine, palmatine, jatrorrhizine, and magnoflorine in P. lavallei D .

Anticancer, cytotoxic potential of Moringa oleifera extracts on HeLa cell line

Shruti Nair, KN Varalakshmi

Journal of Natural Pharmaceuticals 2011 2(3):138-142

Background: The objective of the study was to analyze the anticancer property of Moringa oleifera on HeLa cells and also to analyze its safety on human peripheral lymphocytes. Materials and Methods: Using ethnomedical data approach, the Indian medicinal plant (M. oleifera) that is used in traditional medicine for cancer and non-cancerous diseases was collected. The crude extracts were prepared by alcoholic and aqueous extraction methods using standard protocols. The antiproliferative effects of the aqueous and alcoholic extracts were evaluated in vitro by employing MTT assay, viability test by trypan blue dye exclusion and apoptosis of the cancer cells were confirmed by DNA fragmentation analysis, ethidium bromide- acridine orange (EB/AO) staining. Results: The aqueous extract of M. oleifera showed good cytotoxicity which was concentration dependent. It was contradictory incase of methanolic and hexane extracts, the cell viability was found to increase as the concentration of extract increased. It states that not only the concentration of extract is having an effect on cell viability, even the methods and solvents of extraction are important in exerting their effects on cell lines. At the same time, the extracts showed proliferative effects on the normal human lymphocytes. Conclusion: The aqueous extract of M. oleifera exhibited cytotoxic effects on Hela cells and least cytotoxicity on lymphocytes. For plants that are used as anticancer herbal drugs, our results indicated a correlation between the reported use of these plants and their cytotoxic activity on cancer cells.

Development of the HPLC method to determine the metabolites of isomajdine isolated from Vinca herbacea Waldst. et Kit. growing in Georgia

Natia Gagua, C Aubert, Valentina Vachnadze, Aliosha Bakuridze

Journal of Natural Pharmaceuticals 2011 2(3):163-165

Background: It was decided to develop a reliable HPLC method coupled with ultraviolet detection for simultaneous determination of isomajdine and its metabolites in in vitro experiment. Methods: A liquid liquid extraction was used to extract the compounds from plasma. The analysis was carried out on a ZorbaxC18 column (250mmx4.6mm, 5&#956;m). The mobile phase consisted of water (A) and acetonitril (B) (16:84, V/V) used in gradient mode: 16 (B) for 10 min, 71 (B) for 55 minute, 16 (B) 62 minutes, pumped at 1ml/min. Results: 4 metabolites have been detected and separeted. tha linearity was invetigated. Conclusion: The calibration curves showed good linearity with correlation coefficient greater that 0.996 for the analytes in the investigated range.So the HPLC method is precise, accurate and simple.

Evaluation of the effect of the ethanolic extract of Delonix elata on acute inflammation in rats

K Manimekalai, Kartik J Salwe, Harsha M Shetty

Journal of Natural Pharmaceuticals 2011 2(3):149-153

Background: The ethanolic extract of Delonix elata was studied for its effect on the acute inflammation and comparison of its ulcerogenic potential with ibuprofen. Materials and Methods: The ethanolic extract of the leaves of D. elata was obtained using the Soxhlet apparatus. Swiss albino mice weighing 20-30 g and Wistar albino rats weighing 120-130 g of either sex were used in the study. The antinociceptive property of the plant extract was screened by acetic acid-induced writhing in mice while the anti-inflammatory property was studied using the carrageenan-induced paw edema in rats. The gastric irritant action was tested by administering the drug orally and screening the gastric mucosa macroscopically and microscopically after acute administration of the D. elata extract in rats. The percent inhibition of the writhing response and paw edema was noted. The stomach were excised and opened along the greater curvature. The severity of ulcers was scored from 0 to 5 depending on the size and severity of ulcer. Results: The plant extract at a dose of 100 and 200 mg/kg showed a significant reduction in writhing with 55.56% and 54.76% of inhibition, respectively, and also showed a significant dose-dependent reduction in the volume of paw edema with approximately 55.78% and 66.31% of inhibition, respectively. The histological section after the acute administration of ibuprofen indicated superficial erosion. In contrast, after acute treatment of D. elata, it was found that the surface epithelium was intact. Conclusion: The present results indicate that D. elata may be considered safer than the conventional nonsteroidal anti-inflammatory drugs (NSAIDs) with respect to gastric irritation potential.

Evaluation of hair growth promoting activity of Musa paradisiaca unripe fruit extract

Anil Sidram Savali, Somnath Devidas Bhinge, Hariprasanna R Chitapurkar

Journal of Natural Pharmaceuticals 2011 2(3):120-124

For the evaluation of the hair growth promoting activity of Musa paradisiaca unripe fruit extract, the study was aimed to investigate the hair growth promoting activity of M. paradisiaca unripe fruit extract. Objective: We examined the effect of M. paradisiaca unripe fruit extract for the hair growth promoting activity, which has been traditionally used for treating hair loss. Materials and Methods: The mice were divided into four groups the extract and minoxidil were applied over the shaved skin surface on to the backs of mice and monitored for 30 days. Results: The extract of M. paradisiaca unripe fruit when tested for the hair growth activity was assed by studying hair length and microscopic study of follicles in vehicle control, 2% minoxidil treated and extract treated animals. Conclusion: The findings suggest that extract of M. paradisiaca unripe fruit has potential as a hair growth promoter.

Evaluation of the DPPH radical scavenging activity, total phenols and antioxidant activities in Indian wild Bambusa vulgaris "Vittata" methanolic leaf extract

Arvind Kumar Goyal, Sushil Kumar Middha, Arnab Sen

Journal of Natural Pharmaceuticals 2010 1(1):40-45

Background: Antioxidants have the ability to protect organisms from damage caused by free radical-induced oxidative stress. A lot of research is being carried out worldwide directed toward finding natural antioxidants of plant origin. The antioxidant activity of the methanolic extract of Bambusa vulgaris “Vittata” (BVV) leaves is reported along with screening for photochemical constituents of the Indian, wild BVV methanolic leaf extract. Materials and Methods: The antioxidant activity was tested spectrophotometrically, measuring the ability of the plant extract to scavenge a stable DPPH• free radical and the total phenolic and flavonoid contents. Results: Preliminary studies show the presence of carbohydrates, reducing sugars, flavonoids, steroids, saponins, alkaloids, tannins, anthraquinones and glycosides. The antioxidant activity of the investigated extract has a scavenging ability of hydroxyl peroxide radicals (421.74 ± 25.61 mg/ml) and DPPH

Prominent wound healing properties of indigenous medicines

Nilesh Gupta, UK Jain

Journal of Natural Pharmaceuticals 2010 1(1):2-13

Wound, a clinical entity, is as old as mankind and often possesses problems in clinical practice. Naturally, the investigative curiosity to promote healing continues since ages. A lot of research has been envisaged to develop better healing agents and it has been a challenging task to generate them and keep pace with the problems encountered. Several drugs of plant, mineral, and animal origin are described in the Ayurveda for their wound healing properties. Most of these drugs are derived from plants. Some of these plants have been screened scientifically for the evaluation of their wound healing activity in different pharmacological models. Some Ayurvedic medicinal plants, namely Argemone mexicana, Boerhaavia diffusa, Catharanthus roseas, Diospyros cardifolia, Eclipta alba, Ficus religiosa, Hypericum perforatum, Lawsonia inermis, Merremia tridentate, and Swertia chirata, were found to be effective in experimental models. The rapidity of wound healing depends, to a considerable extent, on the contraction that begins a few days after injury and continues for several weeks. In the present review, attempts are made to understand various aspects of wound healing in terms of percentage closure of wound, period of complete epithelialization, tensile strength, histopathology, and weight of granuloma in different wound models.

Phytosomes in herbal drug delivery

Sunitha Manthena, Prathima Srinivas, Sadanandam

Journal of Natural Pharmaceuticals 2010 1(1):14-18

Phytoconstituents, despite having excellent bioactivity in vitro, demonstrate less or no in vivo actions due to their poor lipid solubility or improper molecular size or both, resulting in poor absorption and poor bioavailability. Lipid solubility and molecular size are the major limiting factors for molecules to pass the biological membrane and to be absorbed systematically following oral or topical administration. Some phytoconstituents are destroyed in the gastric environment when taken orally. The term “phyto” means plant, while “some” means cell-like. Therefore, phytosome is a “phytophospholipid complex” resembling a small cell. Phytosomes are produced by a patented process whereby standardized plant extracts or their constituents are bound to phospholipids, mainly phosphatidylcholine, producing a lipid-compatible molecular complex. Phytosomes exhibit a better pharmacokinetic and pharmacodynamic profile than conventional herbal extracts. The phytosome technology markedly enhances the bioavailability of phytomedicine and has effectively enhanced the bioavailability of many popular herbal extracts, including Milk thistle, Ginkgo biloba, Grape seed, Green tea, Hawthorn, Ginseng etc., and can be developed for various therapeutic uses or dietary supplements.

Isolation of human erythrocyte agglutinins from marine algae

Sudhir Kumar, Urmila Barros

Journal of Natural Pharmaceuticals 2010 1(1):51-54

Marine algae are found in abundance along the shore of Goa, and have not been utilized. This is a report of hemagglutinins isolated from these marine algae. Phosphate-buffered saline extracts from six marine algae were tested for their agglutination activity using human blood cells by a slide assay. The extracts from four species revealed hemagglutinating activity against the tested human erythrocytes. However, the results obtained did not indicate specificity toward any particular human blood group. Further, one extract that showed good agglutination was processed for ammonium sulfate precipitation. The precipitated product was assayed for hemagglutination titer and sugar-binding specificity. The hemagglutination activity of the Sargassum cinnerium extract was observed to be specifically inhibited by one of the tested sugars. Because agglutination is a characteristic of “lectins,” the results obtained are indicative of these marine algae being a rich source of products/novel substances having biotechnological applications.

The in vitro anticancer activity of the crude extract of the sponge-associated fungus Eurotium cristatum and its secondary metabolites

Ana Paula Almeida, Tida Dethoup, Narong Singburaudom, Raquel Lima, Maria Helena Vasconcelos, Madalena Pinto, Anake Kijjoa

Journal of Natural Pharmaceuticals 2010 1(1):25-29

Background: Marine natural products has captivated many researchers over the years and there is always a need for sources of diverse and pharmacologically active leads in the area of anticancer drugs. Materials and Methods: The ethyl acetate extract of the fungus Eurotium cristatum (ECE), isolated from the marine sponge Mycale sp., furnished 2-(2', 3-epoxy-1',3'-heptadienyl)-6-hydroxy-5-(3-methyl-2-butenyl) benzaldehyde (1), 1,8-dihydroxy-6-methoxy-3-methyl-9,10-anthracenedione (physcion,2), and the dioxopiperazine alkaloid echinulin (3). The structures of the compounds were established by Nuclear Magnetic Resonance (NMR) spectral analysis (1H, 13C, DEPT, COSY, HSQC, and HMBC). The ECE and its metabolites were evaluated for their growth inhibitory activity on the following three human tumor cell lines: breast adenocarcinoma (MCF-7), non-small lung cancer (NCI-H460), and melanoma (A375-C5). Results: The results showed that the ECE was active in all the three cell lines, with the values of GI50 = 44.3 ± 1.2, 45.5 ± 7.5, and 71.3 ± 2.1 μg/ml for MCF-7, NCI-H460, and A375-C5, respectively. Compound 1 also exhibited moderate growth inhibitory activity against all the three cell lines (GI50 = 58.3 ± 1.2, 46.0 ± 5.5, and 116.7 ± 7.2 μM for MCF-7, NCI-H460, and A375-C5, respectively), whereas compound 3 showed only weak inhibition against MCF-7 (GI50 = 109.7 ± 0.3 μM) and NCI-H460 (GI50 = 96.7 ± 1.5 μM) but was inactive against A375-C5 (GI50 >150 μM). On the contrary, compound 2 was inactive in all the three cell lines at the highest concentration tested (150 μM). Furthermore, ECE was investigated for its effect on the cell cycle in the NCI-H460 cells. Analysis of the cell cycle profile showed that ECE was able to cause a slight cell arrest in the G1 phase, with a corresponding decrease of cells in the S and G2/M phases. Conclusion: The secondary metabolites isolated [Compound 1] from the crude ethyl acetate extract of the culture of the marine fungus E. cristatum were found as the most potent compound regarding cell growth inhibition.

Antibacterial, antifungal and free radical scavenging activity of Croton gibsonianus Nimm. Grah. (Euphorbiaceae)

KS Vinayaka, D Swathi, TR Prashith Kekuda, K Bhagath, N Mallikarjun

Journal of Natural Pharmaceuticals 2010 1(1):46-50

Background: Croton gibsonianus Nimm. Grah is a shrub belonging to the family Euphorbiaceae and grows in the under-story of the evergreen forests of the Western Ghats. The present study was performed to investigate the antibacterial, antifungal and free radical scavenging potentials of the methanol extract of C. gibsonianus leaves. Materials and Methods: The powdered leaf material was subjected to soxhlet extraction using methanol. The antibacterial and antifungal activities of the methanolic extract were determined by the agar well diffusion method. The free radical scavenging activity was performed using the DPPH free radical scavenging assay. Results: The extract was found to cause marked inhibition of Pseudomonas aeruginosa followed by Escherichia coli and Staphylococcus aureus. Among fungi, Aspergillus niger was inhibited to a greater extent, followed by Chrysosporium indicum, Candida albicans and Trichophyton rubrum. The inhibition of test fungi was dose-dependent. The radical scavenging activity was found to be concentration dependent and the IC 50 value for the extract was found to be 43.78 μg/ml. A phytochemical analysis of the extract showed the presence of saponins, tannins, glycosides and terpenoids. Conclusion: The methanolic extract of C. gibsonianus leaves could be used in the treatment of bacterial and fungal infections and damage caused by free radicals. The presence of various phytochemicals might be responsible for these activities of the extract. Further studies on isolation of constituents from the extract and their biological activities are under investigation.

Potent insecticidal activity of two Streptomyces species isolated from the soils of the western ghats of Agumbe, Karnataka

TR Prashith Kekuda, KS Shobha, R Onkarappa

Journal of Natural Pharmaceuticals 2010 1(1):30-32

Background: The Western ghats, the range of hills running along India's west coast, are well known for their rich and unique assemblage of flora and fauna. The present study was performed to evaluate the insecticidal potential of Actinomycetes isolated from the soils of the Western ghats of Agumbe, Karnataka. Methods: For isolation, the serially diluted soil sample was plated on Starch casein agar and incubated aerobically. The actinomycete isolates were identified by various parameters such as colony morphology, spore arrangement, staining, and biochemical reactions. The isolates were grown in Starch casein broth for seven days, the culture broth was extracted with butanol solvent and concentrated to get crude extract. Insecticidal activity of different concentrations of butanol extract of the isolates was determined against the second instar larvae of Aedes aegypti. The larvicidal effect, in terms of mortality of larvae, of the extracts was determined by counting the number of dead larvae after 24 hours. Results: Two actinomycete isolates were recovered from the soil sample and were identified as the species of Streptomyces on the basis of phenotypic, microscopic, biochemical, and staining characteristics. The colonies of the Streptomyces isolate 1 were creamish-white with yellow pigmentation and the spore arrangement was straight, whereas colonies of the Streptomyces isolate 2 were light grey with dark green pigmentation. The spore arrangement in isolate 2 was of the open loop type. Both the isolates were Gram-positive, non-acid fast, and caused hydrolysis of starch and casein. The insecticidal activity of different concentrations, namely 1.0, 2.5, and 5.0 mg/ml, of butanol extract of the Streptomyces isolates was tested against the second instar larvae of Aedes aegypti. The insecticidal potential of butanol extracts, in terms of larval mortality, was found to be dose dependent. Among the isolates, isolate 2 showed a marked insecticidal activity than isolate 1. At a concentration of 5 mg/ml, both the isolates caused 100% mortality of the larvae. At concentrations of 1 and 2.5 mg/ml, isolate 2 exhibited a stronger larvicidal activity than isolate 1. Conclusion: The insecticidal efficacy of the Streptomyces species might be due to the presence of active constituents in the extract. Isolation and characterization of active constituents from the butanol extract possessing insecticidal potential are to be investigated.

Search
Generic selectors
Exact matches only
Search in title
Search in content
Search in posts
Search in pages
Archives
MonTueWedThuFriSatSun
      1
2345678
9101112131415
16171819202122
23242526272829
3031     
    123
45678910
11121314151617
18192021222324
252627282930 
       
 123456
78910111213
14151617181920
21222324252627
28293031   
       
      1
2345678
9101112131415
16171819202122
23242526272829
30      
   1234
567891011
12131415161718
19202122232425
262728293031 
       
   1234
567891011
12131415161718
19202122232425
262728    
       
1234567
891011121314
15161718192021
22232425262728
293031    
       
    123
45678910
11121314151617
18192021222324
25262728293031
       
  12345
6789101112
13141516171819
20212223242526
27282930   
       
      1
2345678
9101112131415
16171819202122
23242526272829
3031     
    123
45678910
11121314151617
18192021222324
252627282930 
       
 123456
78910111213
14151617181920
21222324252627
28293031   
       
     12
3456789
10111213141516
17181920212223
24252627282930
31      
   1234
567891011
12131415161718
19202122232425
2627282930  
       
1234567
891011121314
15161718192021
22232425262728
293031    
       
     12
3456789
10111213141516
17181920212223
24252627282930
       
  12345
6789101112
13141516171819
20212223242526
2728293031  
       
  12345
6789101112
13141516171819
20212223242526
2728     
       
      1
2345678
9101112131415
16171819202122
23242526272829
3031     
   1234
567891011
12131415161718
19202122232425
262728293031 
       
 123456
78910111213
14151617181920
21222324252627
282930    
       
     12
3456789
10111213141516
17181920212223
24252627282930
31      
   1234
567891011
12131415161718
19202122232425
2627282930  
       
1234567
891011121314
15161718192021
22232425262728
293031    
       
    123
45678910
11121314151617
18192021222324
25262728293031
       
  12345
6789101112
13141516171819
20212223242526
27282930   
       
      1
2345678
9101112131415
16171819202122
23242526272829
3031     
    123
45678910
11121314151617
18192021222324
252627282930 
       
 123456
78910111213
14151617181920
21222324252627
28293031   
       
1234567
891011121314
15161718192021
22232425262728
29      
       
    123
45678910
11121314151617
18192021222324
25262728293031
       
 123456
78910111213
14151617181920
21222324252627
28293031   
       
      1
2345678
9101112131415
16171819202122
23242526272829
30      
   1234
567891011
12131415161718
19202122232425
262728293031 
       
 123456
78910111213
14151617181920
21222324252627
282930    
       
     12
3456789
10111213141516
17181920212223
24252627282930
31      
  12345
6789101112
13141516171819
20212223242526
2728293031  
       
1234567
891011121314
15161718192021
22232425262728
2930     
       
    123
45678910
11121314151617
18192021222324
25262728293031
       
  12345
6789101112
13141516171819
20212223242526
27282930   
       
      1
2345678
9101112131415
16171819202122
23242526272829
3031     
      1
2345678
9101112131415
16171819202122
232425262728 
       
   1234
567891011
12131415161718
19202122232425
262728293031 
       
     12
3456789
10111213141516
17181920212223
24252627282930
       
      1
2345678
9101112131415
16171819202122
23242526272829
30      
   1234
567891011
12131415161718
19202122232425
262728293031 
       
 123456
78910111213
14151617181920
21222324252627
282930    
       
     12
3456789
10111213141516
17181920212223
24252627282930
31      
     12
3456789
10111213141516
17181920212223
2425262728  
       
  12345
6789101112
13141516171819
20212223242526
2728293031  
       
      1
2345678
9101112131415
16171819202122
23242526272829
3031     
    123
45678910
11121314151617
18192021222324
252627282930 
       
 123456
78910111213
14151617181920
21222324252627
28293031   
       
      1
2345678
9101112131415
16171819202122
23242526272829
30      
   1234
567891011
12131415161718
19202122232425
262728293031 
       
1234567
891011121314
15161718192021
22232425262728
293031    
       
  12345
6789101112
13141516171819
20212223242526
2728293031  
       
1234567
891011121314
15161718192021
22232425262728
2930     
       
    123
45678910
11121314151617
18192021222324
25262728293031
       
    123
45678910
11121314151617
18192021222324
25262728   
       
 123456
78910111213
14151617181920
21222324252627
28293031   
       
     12
3456789
10111213141516
17181920212223
24252627282930
31      
   1234
567891011
12131415161718
19202122232425
2627282930  
       
1234567
891011121314
15161718192021
22232425262728
293031    
       
     12
3456789
10111213141516
17181920212223
24252627282930
       
  12345
6789101112
13141516171819
20212223242526
2728293031  
       
      1
2345678
9101112131415
16171819202122
23242526272829
3031     
    123
45678910
11121314151617
18192021222324
252627282930 
       
 123456
78910111213
14151617181920
21222324252627
28293031   
       
      1
2345678
9101112131415
16171819202122
23242526272829
30      
   1234
567891011
12131415161718
19202122232425
262728293031 
       
  12345
6789101112
13141516171819
20212223242526
272829    
       
      1
2345678
9101112131415
16171819202122
23242526272829
3031     
   1234
567891011
12131415161718
19202122232425
262728293031 
       
 123456
78910111213
14151617181920
21222324252627
282930    
       
     12
3456789
10111213141516
17181920212223
24252627282930
31      
   1234
567891011
12131415161718
19202122232425
2627282930  
       
1234567
891011121314
15161718192021
22232425262728
293031    
       
    123
45678910
11121314151617
18192021222324
25262728293031
       
  12345
6789101112
13141516171819
20212223242526
27282930   
       
      1
2345678
9101112131415
16171819202122
23242526272829
3031     
    123
45678910
11121314151617
18192021222324
252627282930 
       
 123456
78910111213
14151617181920
21222324252627
28293031   
       
 123456
78910111213
14151617181920
21222324252627
28      
       
     12
3456789
10111213141516
17181920212223
24252627282930
31      
  12345
6789101112
13141516171819
20212223242526
2728293031  
       
    123
45678910
11121314151617
18192021222324
25262728293031